U.S. patent application number 10/754911 was filed with the patent office on 2004-10-21 for methods and compositions for peptide and protein labeling.
This patent application is currently assigned to Massachusetts Institute of Technology. Invention is credited to Ting, Alice Y..
Application Number | 20040209317 10/754911 |
Document ID | / |
Family ID | 33162068 |
Filed Date | 2004-10-21 |
United States Patent
Application |
20040209317 |
Kind Code |
A1 |
Ting, Alice Y. |
October 21, 2004 |
Methods and compositions for peptide and protein labeling
Abstract
The invention provides compositions and methods of use thereof
for labeling peptide and proteins in vitro or in vivo. The methods
described herein employ biotin ligase mutants and biotin analogs
recognized by such mutants.
Inventors: |
Ting, Alice Y.; (Allston,
MA) |
Correspondence
Address: |
Maria A. Trevisan
Wolf, Greenfield & Sacks, P.C.
600 Atlantic Avenue
Boston
MA
02210
US
|
Assignee: |
Massachusetts Institute of
Technology
Cambridge
MA
|
Family ID: |
33162068 |
Appl. No.: |
10/754911 |
Filed: |
January 9, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60438939 |
Jan 9, 2003 |
|
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Current U.S.
Class: |
435/7.5 ;
530/409 |
Current CPC
Class: |
C12N 9/93 20130101; G01N
33/582 20130101; G01N 2333/9015 20130101 |
Class at
Publication: |
435/007.5 ;
530/409 |
International
Class: |
G01N 033/53 |
Goverment Interests
[0002] This invention was made in part with government support
under grant number K22-HG002671-01 from the National Institutes of
Health. The Government may retain certain rights in the invention.
Claims
What is claimed is:
1. A method for labeling a target protein comprising contacting a
fusion protein with a biotin analog, and allowing sufficient time
for the biotin analog to be conjugated to the fusion protein via an
acceptor peptide, in the presence of a biotin ligase mutant,
wherein the fusion protein is a fusion of the target protein and
the acceptor peptide.
2. The method of claim 1, wherein the biotin analog comprises an
aliphatic carboxylic acid tail.
3. The method of claim 1, wherein the biotin analog comprises a
substitution at a transureido nitrogen (N) of biotin.
4. The method of claim 1, wherein the biotin analog is selected
from the group consisting of an N-ketone biotin analog, a ketone
biotin analog, an N-azide biotin analog, an azide biotin analog, an
N-acyl azide biotin analog, an NBD-GABA biotin analog, a
1,2-diamine biotin analog, an N-alkyne biotin analog and a
tetrathiol biotin analog.
5. The method of claim 1, wherein the biotin analog is
fluorogenic.
6. The method of claim 1, wherein the biotin analog is directly
detectable.
7. The method of claim 6, wherein the biotin analog is coumarin,
fluorescein, rhodamine, rosamine, an Alexa.TM. dye, resorufin,
oregon green, tetramethyl rhodamine, Texas Red.RTM. or BODIPY.
8. The method of claim 1, wherein the biotin analog is labeled with
a directly detectable label.
9. The method of claim 8, wherein the directly detectable label is
selected from the group consisting of a fluorophore, a
radioisotope, a contrast agent, an MRI contrast agent, a PET label,
a phosphorescent label and a luminescent label.
10. The method of claim 1, wherein the biotin analog is labeled
with an indirectly detectable label.
11. The method of claim 10, wherein the indirectly detectable label
is selected from the group consisting of an enzyme, an enzyme
substrate, an antibody, an antibody fragment, an antigen, a hapten,
a ligand, an affinity molecule, a chromogenic substrate, a protein,
a peptide, a nucleic acid, a carbohydrate and a lipid.
12. The method of claim 1, wherein the biotin analog is labeled
with a membrane impermeant label.
13. The method of claim 1, wherein the biotin analog is labeled
after conjugation to the fusion protein.
14. The method of claim 1, wherein the biotin analog is labeled
with a singlet oxygen radical generator.
15. The method of claim 14, wherein the singlet oxygen generator is
resorufin, malachite green, fluorescein or diaminobenzidine.
16. The method of claim 1, wherein the biotin analog is labeled
with an analyte-binding group.
17. The method of claim 16, wherein the analyte-binding group is a
metal chelator.
18. The method of claim 17, wherein the metal chelator is EDTA,
EGTA, a pyridinium, an imidazole or a thiol.
19. The method of claim 1, wherein the biotin analog is labeled
with a heavy atom carrier.
20. The method of claim 19, wherein the heavy atom carrier is
iodine.
21. The method of claim 1, wherein the biotin analog is labeled
with an affinity tag.
22. The method of claim 21, wherein the affinity tag is selected
from the group consisting of a histidine tag, a GST tag, a FLAG tag
and an HA tag.
23. The method of claim 1, wherein the biotin analog is labeled
with a photoactivatable cross-linker.
24. The method of claim 23, wherein the photoactivatable
cross-linker is selected from the group consisting of benzophenones
and aziridines.
25. The method of claim 1, wherein the biotin analog is labeled
with a photoswitch label.
26. The method of claim 25, wherein the photoswitch label is an
azobenzene.
27. The method of claim 1, wherein the biotin analog is labeled
with a photolabile protecting group.
28. The method of claim 27, wherein the photolabile protecting
group is a nitrobenzyl group, a dimethoxy nitrobenzyl group or
NVOC.
29. The method of claim 1, wherein the biotin analog is labeled
with a peptide comprising non-naturally occurring amino acids.
30. The method of claim 1, wherein the target protein is a cell
surface protein.
31. The method of claim 1, wherein the fusion protein is in a
cell.
32. The method of claim 31, wherein the cell expresses the biotin
ligase mutant.
33. The method of claim 31, wherein the cell is a eukaryotic
cell.
34. The method of claim 31, wherein the cell is a bacterial
cell.
35. The method of claim 33, wherein the eukaryotic cell is a
mammalian cell, a Drosophila cell, a Zebrafish cell, a Xenopus
cell, a yeast cell or a C. elegans cell.
36. The method of claim 1, wherein the acceptor peptide comprises
an amino acid sequence of SEQ ID NO: 4.
37. The method of claim 1, wherein the acceptor peptide comprises
an amino acid sequence of SEQ ID NO: 5.
38. The method of claim 1, wherein the acceptor peptide is N- or C-
terminally fused to the target protein.
39. The method of claim 1, wherein the biotin ligase mutant has an
amino acid substitution at 83, 89, 90, 91, 92, 107, 112, 115, 116,
117, 118, 123, 132, 134, 142, 186, 188, 189, 190, 204, 206, 207
and/or 235.
40. The method of claim 39, wherein the amino acid substitution is
at T90, C107, Q112, G115, Y132, S134, V189 and/or I207.
41. The method of claim 40, wherein the amino acid substitution is
at T90.
42. The method of claim 41, wherein the amino acid substitution is
selected from the group consisting of T90G, T90A and T90V.
43. The method of claim 42, wherein the amino acid substitution is
T90G.
44. The method of claim 43, wherein the biotin analog is N-ketone
biotin analog.
45. The method of claim 43, wherein the biotin ligase mutant has an
amino acid sequence of SEQ ID NO: 6.
46. The method of claim 41, wherein the biotin ligase mutant
further comprises an amino acid substitution at N91.
47. The method of claim 46, wherein the amino acid substitution at
N91 is N91S, N91G, N91A or N91L.
48. The method of claim 47, wherein the biotin ligase mutant
comprises amino acid substitutions of T90G and N91S.
49. The method of claim 48, wherein the biotin analog is N-alkyne
biotin analog.
50. The method of claim 48, wherein the biotin ligase mutant has an
amino acid sequence of SEQ ID NO: 7.
51. The method of claim 1, wherein the biotin ligase mutant
comprises amino acid substitutions of T90G/N91G, T90A/N91A or
T90A/N91L.
52. The method of claim 39, wherein the amino acid substitution is
C 107G, Q112M, G115A, Y132G, Y132A, S134G, V189G and/or I207S.
53. The method of claim 1, wherein the method is performed in a
cell free environment.
54. The method of claim 1, wherein the method is performed in a
cell.
55. The method of claim 1, wherein the method is performed in a
subject.
56. The method of claim 1, wherein the acceptor peptide is fused to
the target protein via a cleavable bond or linker.
57. A composition comprising a biotin ligase mutant that binds to a
biotin analog.
58.-80. (Canceled)
81. A composition comprising a nucleic acid encoding a biotin
ligase mutant comprising an amino acid substitution at 83, 89, 90,
91, 92, 107, 112, 115, 116, 117, 118, 123, 132, 134, 142, 186, 188,
189, 190, 204, 206, 207 and/or 235.
82.-94. (Canceled)
95. A composition comprising a biotin analog that binds to a biotin
ligase mutant, wherein the biotin analog is alkylated at a
trans-ureido nitrogen (N) of biotin.
96.-109. (Canceled)
110. A composition comprising a biotin analog that binds to a
biotin ligase mutant, wherein the biotin analog is ketone biotin
analog or NBD-GABA.
111. A phage display library comprising a biotin ligase mutant
having an amino acid substitution at 83, 89, 90, 91, 92, 107, 112,
115, 116, 117, 118, 123, 132, 134, 142, 186, 188, 189, 190, 204,
206, 207 or 235.
112.-121. (Canceled)
122. A method for identifying a biotin ligase mutant having
specificity for a biotin analog comprising contacting a biotin
analog with an acceptor peptide in the presence of a candidate
biotin ligase mutant molecule, and detecting a biotin analog that
is bound to the acceptor peptide, wherein the presence of a biotin
analog bound to an acceptor peptide indicates that the candidate
biotin ligase mutant molecule is a biotin ligase mutant having
specificity for a biotin analog.
123.-145. (Canceled)
146. A method for identifying a biotin analog having specificity
for a biotin ligase mutant comprising combining an acceptor peptide
with a labeled biotin in the presence of a biotin ligase mutant and
determining a control level of biotin incorporation, combining an
acceptor peptide with a labeled biotin and a candidate biotin
analog molecule in the presence of a biotin ligase mutant and
determining a test level of biotin incorporation, and comparing the
control and test levels of biotin incorporation, wherein a test
level that is less than a control level is indicative of a biotin
analog having specificity for a biotin ligase mutant.
147.-161. (Canceled)
Description
RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. 119(e) to
U.S. Provisional Patent Application No. 60/438,939, filed Jan. 9,
2003, entitled "SITE-SPECIFIC LABELING OF RECOMBINANT PROTEINS IN
LIVING CELLS WITH ENGINEERED FLUOROPHORE TRANSFERASE", the entire
contents of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] To track protein expression, localization, or conformational
changes as components of cellular signaling pathways, biologists
need general tools for the in vivo site-specific labeling of
proteins with fluorophores or other useful probes. Traditional
chemical methods rely on the nucleophilicity of cysteine or lysine
side chains and are too promiscuous for in vivo use, and genetic
methods such as fusion to green fluorescent protein (GFP) carry
bulky payloads (GFP is 238 amino acids) and are limited in the
color range and nature of the spectroscopic readout.
[0004] A survey of the existing methods for targeting small
molecules to protein sequences reveals that the shorter the target
sequence, the less specific the conjugation chemistry. For
instance, very specific conjugation can be achieved by fusing the
protein O.sup.6-alkylguanine-DN- A alkyltransferase (AGT) to the
target protein of interest, and then adding a fluorescently-labeled
O.sup.6-benzylguanine suicide substrate for the AGT. (Keppler, A.
et al. Nat. Biotechnol. 21, 86-89, 2003). However, the AGT tag is
207 amino acids and introduces a large amount of steric bulk.
Smaller peptide tags are more desirable, but difficult to target
with small molecules with high specificity. For example, cysteine
labeling is not at all specific inside cells, and tetracysteine
labeling (Griffin, B A et al. Science 281, 269-272, 1998), while
much better, is still insufficiently specific for most applications
and allows only a small set of probes to be introduced.
Transglutaminase is already used to label glutamine side chains
with fluorophores in vitro (Sato, H. et al. Biochemistry 35,
13072-13080, 1996), however it is relatively promiscuous for
peptide and protein substrates, precluding its use in mammalian
cells. In vitro labeling and microinjection has the disadvantage
that protein localization and abundance may be altered.
Polyhistidine tag methodology has the disadvantage that nickel is
toxic, promiscuous, membrane impermeant and a quencher of
fluorescence.
[0005] Accordingly, there exists a need for a method to label
proteins and peptides that is specific and which offers a variety
of a labeling options.
SUMMARY OF THE INVENTION
[0006] The invention relates in part to labeling of proteins (or
fragments thereof) using biotin ligase mutants. The methods and
compositions provided by the invention provide labeling specificity
while also expanding the scope of compatible probe structures for
labeling of proteins. Labeling of peptides or proteins can be
performed in vitro or in vivo. The invention also provides, inter
alia, biotin ligase mutants and biotin analogs and methods of use
thereof for labeling proteins. It also provides screening methods
for identifying further biotin ligase mutants and biotin
analogs.
[0007] Thus, in one aspect, the invention provides a method for
labeling a target protein comprising contacting a fusion protein
with a biotin analog, and allowing sufficient time for the biotin
analog to be conjugated to the fusion protein via an acceptor
peptide, in the presence of a biotin ligase mutant, wherein the
fusion protein is a fusion of the target protein and the acceptor
peptide.
[0008] Various embodiments apply equally to this and other aspects
of the invention. These are discussed below.
[0009] In one embodiment, the biotin analog may comprise an
aliphatic carboxylic acid tail. In another embodiment and
potentially additionally, the biotin analog may comprise an amino
acid substitution at a trans-ureido nitrogen (N) of biotin.
Examples of biotin analogs include but are not limited to N-ketone
biotin analog, a ketone biotin analog, an N-azide biotin analog, an
azide biotin analog, an N-acyl azide biotin analog, an NBD-GABA
biotin analog, a 1,2-diamine biotin analog, an N-alkyne biotin
analog and a tetrathiol biotin analog. The biotin analog may be
fluorogenic. Alternatively, the biotin analog may be directly
detectable. Examples include but are not limited to coumarin,
fluorescein, rhodamine, rosamine, an Alexa.TM. dye, resorufin,
oregon green, tetramethyl rhodamine, Texas Red.RTM. and BODIPY. In
still other embodiments, the biotin analog is labeled with a
directly detectable label, such as but not limited to fluorophore,
a radioisotope, a contrast agent, an MRI contrast agent, a PET
label, a phosphorescent label and a luminescent label.
Alternatively, the biotin analog is labeled with an indirectly
detectable label such as but not limited to an enzyme, an enzyme
substrate, an antibody, an antibody fragment, an antigen, a hapten,
a ligand, an affinity molecule, a chromogenic substrate, a protein,
a peptide, a nucleic acid, a carbohydrate and a lipid. In still a
further embodiment, the biotin analog is labeled with a membrane
impermeant label.
[0010] The biotin analog may be labeled before or after conjugation
to the fusion protein. In one embodiment, the acceptor peptide is
fused to the target protein via a cleavable bond or linker.
[0011] The biotin analog may be labeled with a variety of labels,
described herein. For example, the biotin analog may be labeled
with a singlet oxygen radical generator such as but not limited to
resorufin, malachite green, fluorescein or diaminobenzidine. The
biotin analog may be labeled with an analyte-binding group, such as
a metal chelator, non-limiting examples of which include EDTA,
EGTA, a pyridinium, an imidazole and a thiol. The biotin analog may
be labeled with a heavy atom carrier, such as but not limited to
iodine. The biotin analog may be labeled with an affinity tag such
as but not limited to a histidine tag, a GST tag, a FLAG tag and an
HA tag. The biotin analog may be labeled with a photoactivatable
cross-linker such as but not limited to benzophenones and
aziridines. The biotin analog may be labeled with a photoswitch
label such as but not limited to azobenzene. The biotin analog may
be labeled with a photolabile protecting group such as but not
limited to a nitrobenzyl group, a dimethoxy nitrobenzyl group or
NVOC. The biotin analog may be labeled with a peptide comprising
non-naturally occurring amino acids, examples of which are provided
herein.
[0012] The target protein may be a cell surface protein, or an
intracellular protein but it is not so limited. In one embodiment,
the fusion protein is in a cell. Depending upon the method, the
biotin ligase mutant may be expressed by a cell (for example the
cell harboring the fusion protein) or it may be added to a protein
in a cell free environment. In one embodiment, the cell is a
eukaryotic cell while in another it is a bacterial cell. Examples
of eukaryotic cell include but are not limited to a mammalian cell,
a Drosophila cell, a Zebrafish cell, a Xenopus cell, a yeast cell
or a C. elegans cell.
[0013] In one embodiment, the acceptor peptide comprises an amino
acid sequence of SEQ ID NO: 4. In another embodiment, the acceptor
peptide comprises an amino acid sequence of SEQ ID NO: 5. The
acceptor peptide may be N- or C-terminally fused to the target
protein.
[0014] In still another embodiment, the biotin ligase mutant has an
amino acid substitution at 83, 89, 90, 91, 92, 107, 112, 115, 116,
117, 118, 123, 132, 134, 142, 186, 188, 189, 190, 204, 206, 207 or
235. In some embodiments, the amino acid substitution is at T90,
C107, Q112, G115, Y132, S134, V189 or I207. In some important
embodiments, the amino acid substitution is at T90 and includes but
is not limited to T90G, T90A and T90V. In a particular embodiment,
the amino acid substitution is at T90G and optionally the biotin
analog is N-ketone biotin analog. The biotin ligase mutant may
further comprise an amino acid substitution at N91 such as but not
limited to N91S, N91G, N91A or N91L. In a particular embodiment,
the biotin ligase mutant comprises amino acid substitutions of T90G
and N91S. In a related embodiment, the biotin analog is N-alkyne
biotin analog. In still other embodiments, the biotin ligase mutant
comprises amino acid substitutions of T90G/N91G, T90A/N90A or
T90A/N91L. In still other embodiments, the amino acid substitution
is C107G, Q112M, G115A, Y132G, Y132A, S134G, V189G or I207S. The
biotin ligase mutant may have an amino acid sequence of SEQ ID NO:
6 or SEQ ID NO: 7.
[0015] The method may be performed in a cell free environment or it
may be performed in the context of a cell (e.g., in a cell or on a
cell). The method may also be performed in a subject.
[0016] In another aspect, the invention provides a composition
comprising a biotin ligase mutant that binds to a biotin analog. In
one embodiment, the biotin ligase mutant comprises an amino acid
substitution in a biotin interaction and activation domain. All of
the foregoing embodiments relating to biotin ligase mutants and
biotin analogs also apply to this aspect of the invention and thus
will not be recited again. In another embodiment, the biotin ligase
mutant is isolated. The biotin ligase mutant may have reduced
binding affinity to biotin. In another embodiment, the biotin
ligase mutant has wild type binding affinity to biotin.
[0017] In still another aspect, the invention provides a
composition comprising a nucleic acid encoding a biotin ligase
mutant comprising an amino acid substitution at 83, 89, 90, 91, 92,
107, 112, 115, 116, 117, 118, 123, 132, 134, 142, 186, 188, 189,
190, 204, 206, 207 or 235. As used herein, the amino acid positions
recited herein are relative to the wild type biotin ligase having
an amino acid sequence as shown in SEQ ID NO:2. It is to be
understood that the biotin ligase mutant may comprise one or more
of the aforementioned amino acid substitutions. In particular
embodiments, the amino acid substitution is selected from the group
consisting of T90G, T91A, T90V, N91S, N91G, N91A, N91L, C 107G,
Q112M, Q112G, G115A, Y132G, Y132A, S134G, V189G, and I207S. The
nucleic acid is preferably isolated, but it is not so limited. In
some embodiments, the nucleic acid is inducibly expressed. The
nucleic acid may encode any of the biotin ligase mutants described
herein. The invention further provides vectors that comprise
nucleic acid that encode any of the biotin ligase mutants described
herein and host cells that comprise these vectors. The invention
further provides a process for preparing a biotin ligase mutant
comprising culturing the host cells described herein and recovering
the biotin ligase mutant from the culture.
[0018] In yet another aspect, the invention provides a composition
comprising a biotin analog that binds to a biotin ligase mutant,
wherein the biotin analog is alkyated at a trans-ureido nitrogen
(N) of biotin. Examples of such biotin analogs include but are not
limited to an N-ketone biotin analog, an N-azide biotin analog, an
N-acyl azide biotin analog, and an N-alkyne biotin analog. In one
embodiment, the biotin analog is not recognized by wild type biotin
ligase. In another embodiment, the biotin analog is isolated. Other
embodiments relating to biotin analogs and biotin ligase mutants
are recited herein.
[0019] In another aspect, the invention provides a composition
comprising a biotin analog that binds to a biotin ligase mutant,
wherein the biotin analog is ketone biotin analog or NBD-GABA.
[0020] In still another aspect, the invention provides a phage
display library comprising a biotin ligase mutant having an amino
acid substitution at 83, 89, 90, 91, 92, 107, 112, 115, 116, 117,
118, 123, 132, 134, 142, 186, 188, 189, 190, 204, 206, 207 or 235.
In one embodiment, the amino acid substitution is at T90, G115,
Y132, C107, Q112, V189, I207 or S134. In another embodiment, the
amino acid substitution is at T90 and may be but is not limited to
T90G, T90A or T90V. In another embodiment, the biotin ligase mutant
further comprises an amino acid substitution at N91 such as but not
limited to N91S, N91G, N91A or N91L. In one embodiment, the biotin
ligase mutant comprises amino acid substitutions of T90G and N91S.
In another embodiment, it comprises one or more of the amino acid
substitutions of C107G, Q112M, G115A, Y132G, Y132A, V189G, S134G,
I207S, T90G/N91G, T90A/N91A and T90A/N91L. The amino acid
substitution may be at 90, 91, 112, 115, 116, 132 or 188. In a
particular embodiment, the library has at least about
1.times.10.sup.8 or about 1.times.10.sup.9 members.
[0021] In still another aspect, the invention provides a method for
identifying a biotin ligase mutant having specificity for a biotin
analog comprising contacting a biotin analog with an acceptor
peptide in the presence of a candidate biotin ligase mutant
molecule, and detecting a biotin analog that is bound to the
acceptor peptide, wherein the presence of the biotin analog bound
to the acceptor peptide indicates that the candidate biotin ligase
mutant molecule is a biotin ligase mutant having specificity for
the biotin analog. The candidate molecule may be a library member
such as but not limited to a phage display library member. In one
embodiment, the candidate molecule is bound to a solid support
while in another it is soluble. Various embodiments of biotin
analog are possible as recited herein. In one embodiment, detecting
a biotin analog comprises detecting the detectable label conjugated
to the biotin analog. The acceptor peptide may have an amino acid
sequence comprising SEQ ID NO: 4 or SEQ ID NO: 5, but it is not so
limited. In one embodiment, the biotin analog is detected using an
antibody. The biotin analog may be detected using a detection
system such as but not limited to fluorescent detection system, a
luminescent detection system, a photographic film detection system,
an enzyme detection system, an electron spin resonance detection
system, a scanning tunneling microscopy (STM) detection system, an
optical detection system and a nuclear magnetic resonance (NMR)
detection system.
[0022] In one embodiment, the method further comprises removing
unbound biotin analog prior to detecting bound biotin analog. The
method may also further comprise identifying a biotin ligase mutant
having specificity for a biotin analog and biotin. In a related
embodiment, the biotin ligase mutant having specificity for a
biotin analog and biotin is identified by contacting biotin with an
acceptor peptide in the presence of a candidate molecule, and
detecting biotin that is bound to the acceptor peptide, wherein the
presence of biotin bound to an acceptor peptide indicates that the
candidate molecule is a biotin ligase mutant having specificity for
a biotin analog and biotin.
[0023] The method may also further comprise isolating the candidate
molecule that is a biotin ligase mutant having specificity for a
biotin analog or the biotin ligase mutant having specificity for a
biotin analog and biotin.
[0024] In another aspect, the invention provides a method for
identifying a biotin analog having specificity for a biotin ligase
mutant comprising combining an acceptor peptide with a labeled
biotin in the presence of a biotin ligase mutant and determining a
control level of biotin incorporation, combining an acceptor
peptide with a labeled biotin and a candidate biotin analog
molecule in the presence of a biotin ligase mutant and determining
a test level of biotin incorporation, and comparing the control and
test levels of biotin incorporation, wherein a test level that is
less than a control level is indicative of a biotin analog having
specificity for a biotin ligase mutant. Various embodiments
relating to the biotin ligase mutant, the biotin analog and the
acceptor peptide are recited above.
[0025] These and other objects of the invention will be described
in further detail in connection with the detailed description of
the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1A shows biotinylation of the lysine side chain of the
consensus peptide sequence of biotin ligase (BirA). (Chapman-Smith
et al. J. Nutr. 129, 477S-484S, 1999).
[0027] FIG. 1B shows the structures of biotin as well as various
biotin analogs. NBD-GABA (7-nitrobenz-2-oxa-1,3-diazole
.gamma.-aminobutyric acid) is a fluorophore with a similar size and
shape to biotin. Biotin isostere (labeled as ketone) has a
bio-orthogonal ketone functionality that can be chemoselectively
modified with hydrazine- and alkoxyamine-derivatized probes as
shown in FIG. 2. (Cornish et al. J. Am. Chem. Soc. 118, 8150-8151,
1996; Mahal et al. Science 276, 1125-1128, 1997.) Coumarin and
fluorescein are directly detectable biotin analogs.
[0028] FIG. 2 shows the labeling of biotin analogs with labels.
Biotin analogs that introduce unique chemical handles for
subsequent modification by a range of probes in the live cell
context are shown. "F" represents any fluorophore. The ketone
biotin analog can be selectively conjugated to hydrazide,
hydroxylamino, and thiosemicarbazide groups under physiological
conditions. The azide biotin analog can be selectively coupled to
phosphines via the modified Staudinger reaction. (Saxon and
Bertozzi, Science 287:2007-2010, 2000.) The tetrathiol biotin
analog can form a stable adduct with the fluorescein-arsenic
derivative (FlAsH) shown. The reaction of azide with a fluorogenic
biotin analog (e.g., non-fluorescent coumarin phosphine) results in
a detectable compound (e.g., fluorescent coumarin).
[0029] FIG. 3A shows a phage display scheme to select for desired
biotin ligase mutants from a library. Wild type biotin ligase has
already been successfully displayed on phage and enriched in model
selections by Neri et al. (Heinis et al. Protein Engineering
14:1043-1052, 2001.)
[0030] FIG. 3B shows the results of biotinylation activity assays
for wild type biotin ligase in soluble or phage displayed form, and
either in the presence or absence of ATP.
[0031] FIG. 4 shows a synthesis pathway for the ketone biotin
analog.
[0032] FIG. 5 shows a synthesis pathway for the N-acyl azide and
NBD-GABA biotin analogs.
[0033] FIG. 6A shows expression of wild type biotin ligase and
biotin ligase mutants.
[0034] FIG. 6B shows the results of biotinylation activity assays
for various biotin ligase mutants. The biotin ligase mutants
harboring amino acid substitutions of T90G, G115A or T90V have
affinity for biotin comparable to wild type biotin ligase.
[0035] FIG. 7 shows the alignment of the amino acid (SEQ ID NO: 1)
and nucleotide (SEQ ID NO: 2) sequence of wild type biotin
ligase.
BRIEF DESCRIPTION OF THE SEQUENCE LISTING
[0036] SEQ ID NO: 1 is the amino acid sequence of wild type biotin
ligase.
[0037] SEQ ID NO: 2 is the nucleotide sequence of wild type biotin
ligase.
[0038] SEQ ID NO: 3 is a consensus amino acid sequence of an
acceptor peptide.
[0039] SEQ ID NO: 4 is the amino acid sequence of a 13 amino acid
acceptor peptide.
[0040] SEQ ID NO: 5 is the amino acid sequence of an acceptor
peptide (AviTag.TM.).
[0041] SEQ ID NO: 6 is the amino acid sequence of a biotin ligase
mutant having a T90G amino acid substitution.
[0042] SEQ ID NO: 7 is the amino acid sequence of a biotin ligase
mutant having T90G and N91S amino acid substitutions.
[0043] SEQ ID NO: 8 is the amino acid sequence of a biotin ligase
mutant having possible amino acid substitutions at amino acid
positions 83, 89, 90, 91, 92, 107, 112, 115, 116, 117, 118, 123,
132, 134, 142, 186, 188, 189, 190, 204, 206, 207, or 235.
[0044] SEQ ID NO: 9 is the amino acid sequence of a biotin ligase
mutant having T90G, T90A, or T90V amino acid substitutions.
[0045] SEQ ID NO: 10 is the amino acid sequence of a biotin ligase
mutant having T90G, T90A, or T90V and N91S, N91G, N91A, or N91L
amino acid substitutions.
[0046] SEQ ID NO: 11 is the amino acid sequence of a biotin ligase
mutant having T90G and N91G amino acid substitutions.
[0047] SEQ ID NO: 12 is the amino acid sequence of a biotin ligase
mutant having T90A and N91A amino acid substitutions.
[0048] SEQ ID NO: 13 is the amino acid sequence of a biotin ligase
mutant having T90A and N91L amino acid substitutions.
[0049] SEQ ID NO: 14 is the amino acid sequence of a biotin ligase
mutant having C107G amino acid substitution.
[0050] SEQ ID NO: 15 is the amino acid sequence of a biotin ligase
mutant having Q112M amino acid substitution.
[0051] SEQ ID NO: 16 is the amino acid sequence of a biotin ligase
mutant having G115A amino acid substitution.
[0052] SEQ ID NO: 17 is the amino acid sequence of a biotin ligase
mutant having Y132G amino acid substitution.
[0053] SEQ ID NO: 18 is the amino acid sequence of a biotin ligase
mutant having Y132A amino acid substitution.
[0054] SEQ ID NO: 19 is the amino acid sequence of a biotin ligase
mutant having S143G amino acid substitution.
[0055] SEQ ID NO: 20 is the amino acid sequence of a biotin ligase
mutant having V189G amino acid substitution.
[0056] SEQ ID NO: 21 is the amino acid sequence of a biotin ligase
mutant having I207S amino acid substitution.
DETAILED DESCRIPTION OF THE INVENTION
[0057] The invention relates to protein labeling in vivo and in
vitro. Prior attempts to label specific proteins have been
frustrated by a lack of reagents with sufficient specificity. The
invention aims to overcome this lack of specificity through the use
of biotin ligase mutants and biotin analogs that are recognized by
such mutants.
[0058] The invention therefore provides, inter alia, methods for
labeling proteins in vitro or in vivo. The method generally
involves contacting a biotin analog with a fusion protein in the
presence of a biotin ligase mutant, and allowing sufficient time
for conjugation of the biotin analog to the fusion protein. Times
and reaction conditions suitable for biotin ligase mutant activity
will generally be comparable to those for wild type biotin ligase
which are known in the art. (See for example Examples herein and
Avidity technical literature.)
[0059] The various components of this reaction will be described in
greater detail herein. Briefly, the fusion protein is a fusion of
the target protein (i.e., the protein which is to be labeled) and
an acceptor peptide (i.e., the peptide sequence that acts as a
substrate for the biotin ligase mutant). If the method is performed
in vivo, the nucleic acid sequence encoding the fusion protein will
be introduced into the cell and transcription and translation
allowed to occur. If the method is performed in vitro, the fusion
protein will simply be added to the reaction mixture.
[0060] As used herein, protein labeling in vitro means labeling of
a protein in a cell free environment. As an example, such a protein
can be combined with a biotin ligase mutant and a biotin analog
under appropriate conditions and thereby labeled, in for example a
test tube or a well of a multiwell plate.
[0061] As used herein, protein labeling in vivo means labeling of a
protein in the context of a cell. The method can be used to label
proteins that are intracellular proteins or cell surface proteins.
The cell may be present in a subject (e.g., an insect such as
Drosophila, a rodent such as a mouse, a human, and the like) or it
may be present in culture.
[0062] The biotin ligase mutant may also be expressed by the cell
in some instances. In other instances, however, the biotin ligase
mutant may simply be added to the reaction mixture (if in vitro) or
to the cell (if the target protein is a cell surface protein and
the acceptor peptide is located on the extracellular domain of the
target protein).
[0063] According to the method, the biotin ligase mutant conjugates
the biotin analog to the acceptor peptide that is fused (either at
the nucleic acid level or post-translationally) to the target
protein. The method is independent of the protein type and thus any
protein can be labeled in this manner. The product of this labeling
reaction may or may not be directly detectable however depending
upon the nature of the biotin analog, as described herein.
Accordingly, it may be necessary to react the conjugated biotin
analog with a detectable label. If the method is performed in vivo,
the detectable label is preferably one capable of diffusion into a
cell. If the method is used to label a cell surface protein, then
preferably the biotin analog is labeled with a membrane impermeant
label in order to reduce entry and accumulation of the label
intracellularly. The biotin analog may be labeled prior to or after
conjugation to the fusion protein.
[0064] Labeling of proteins allows one to track the movement and
activity of such proteins. It also allows cells expressing such
proteins to be tracked and imaged, as the case may be. The methods
can be used in cells from virtually any organism including insect,
yeast, frog, worm, fish, rodent, human and the like.
[0065] The method can be used to label virtually any protein.
Examples include but are not limited to signal transduction
proteins (e.g., cell surface receptors, kinases, adapter proteins),
nuclear proteins (transcription factors, histones), mitochondrial
proteins (cytochromes, transcription factors) and hormone
receptors.
[0066] Biotin ligase (BirA) is an 321 amino acid, 33.5 kD enzyme
derived from E. coli that catalyzes the context-specific
conjugation of biotin to a lysine .epsilon.-amine in biotin
retention and biosynthesis pathways, as shown in FIG. 1A. This
reaction is ATP-dependent. As used herein, wild type biotin ligase
refers to a naturally occurring bacterial biotin ligase having wild
type biotinylation activity. SEQ ID NO: 1 represents the amino acid
sequence of wild type biotin ligase (GenBank Accession No. M10123).
SEQ ID NO: 2 represents the nucleotide sequence of wild type biotin
ligase (GenBank Accession No. M10123).
[0067] Biotin ligase is also known as biotin protein ligase, biotin
operon repressor protein, BirA, biotin holoenzyme synthetase and
biotin-[acetyl-CoA carboxylase] synthetase.
[0068] The reaction between biotin ligase and its substrate
(discussed below) is referred to as orthogonal. This means that
neither the ligase nor its substrate react with any other enzyme or
molecule when present either in their native environment (i.e., a
bacterial cell) or more importantly for the purposes of the
invention in a non-native environment (e.g., a mammalian cell).
Accordingly, the invention takes advantage of the high degree of
specificity which has evolved between biotin ligase and its
substrate.
[0069] The only known natural substrate in bacteria of wild type
biotin ligase is lysine 122 of the biotin carboxyl carrier protein
(BCCP). Chapman-Smith et al. J. Nutr. 129:477S-484S, 1999.) A 13-15
amino acid minimal substrate sequence encompassing lysine 122 has
been identified as the minimal peptide recognition sequence for
biotin ligase. As used herein, an "acceptor peptide" is a protein
or peptide having an amino acid sequence that is a substrate for a
biotin ligase mutant (i.e., a biotin ligase mutant recognizes and
is capable of conjugating a biotin analog or biotin to the
peptide). The acceptor peptide may have an amino acid sequence of
Leu Xaa.sub.1 Xaa.sub.2 Ile Xaa.sub.3 Xaa.sub.4 Xaa.sub.5 Xaa.sub.6
Lys Xaa.sub.7 Xaa.sub.8 Xaa.sub.9 Xaa.sub.10 (SEQ. ID NO:3), where
Xaa.sub.1 is any amino acid, Xaa.sub.2 is any amino acid other than
large hydrophobic amino acids (such as Leu, Val, Ile, Trp, Phe,
Tyr); Xaa.sub.3 is Phe or Leu, Xaa4 is Glu or Asp; Xaa.sub.5 is
Ala, Gly, Ser, or Thr; Xaa.sub.6 is Gln or Met; Xaa.sub.7 is Ile,
Met, or Val; Xaa.sub.8 is Glu, Leu, Val, Tyr, or Ile; Xaa.sub.9 is
Trp, Tyr, Val, Phe, Leu, or Ile; and Xaa.sub.10 is preferably Arg
or His but may be any amino acid other than acidic amino acids such
as Asp or Glu. Acceptor peptides are known in the art and examples
are described in U.S. Pat. Nos. 5,723,584; 5,874,239 and 5,932,433,
the entire contents of which are herein incorporated by reference.
In important embodiments, the acceptor peptide comprises the amino
acid sequence of LNDIFEAQKIEWH (SEQ ID NO: 4). In another
embodiment, the acceptor peptide comprises an amino acid sequence
of GLNDIFEAQKIEWHE (SEQ ID NO: 5). Acceptor peptides can be
synthesized using standard peptide synthesis techniques. They are
also commercially available under the trade name AviTag.TM. from
Avidity (Boulder, Colo.).
[0070] The acceptor peptide is used in the methods of the invention
to tag target proteins that are to be labeled by biotin ligase
mutants. The acceptor peptide and target protein may be fused to
each other either at the nucleic acid or amino acid level.
Recombinant DNA technology for generating fusion nucleic acids that
encode both the target protein and the acceptor peptide are known
in the art. Additionally, the acceptor peptide may be fused to the
target protein post-translationally. Such linkages may include
cleavable linkers or bonds which can be cleaved once the desired
labeling is achieved. Such bonds may be cleaved by exposure to a
particular pH, or energy of a certain wavelength, and the like.
Cleavable linkers are known in the art. Examples include
thiol-cleavable cross-linker 3,3'-dithiobis(succinimidyl
proprionate), amine-cleavable linkers, and succinyl-glycine
spontaneously cleavable linkers.
[0071] The acceptor peptide can be fused to the target protein at
any position. In some instances, it is preferred that the fusion
not interfere with the activity of the target protein, accordingly,
the acceptor peptide is fused to the protein at positions that do
not interfere with the activity of the protein. Generally, the
acceptor peptides can be C- or N-terminally fused to the target
proteins. In still other instances, it is possible that the
acceptor peptide is fused to the target protein at an internal
position (e.g., a flexible internal loop). These proteins are then
susceptible to specific tagging by biotin ligase and biotin ligase
mutants in vivo and in vitro. This specificity is possible because
neither biotin ligase nor the acceptor peptide react with any other
enzymes or peptides in a cell.
[0072] Thus, the invention is directed in part to generating biotin
ligase mutants that recognize biotin analogs and conjugate such
analogs to the acceptor peptide. Biotin ligase mutants can be
generated in any number of ways, including phage display
technology, described in greater detail herein.
[0073] The labeling methods of the invention rely on the activity
of biotin ligase mutants that recognize and conjugate biotin
analogs onto fusion proteins via the acceptor peptide. The
invention provides biotin ligase mutants that recognize biotin
analogs, and in some instances, biotin itself. As used herein, a
biotin ligase mutant is a variant of biotin ligase that is
enzymatically active towards a biotin analog (such as those
described herein). As used herein, "enzymatically active" means
that the mutant is able to recognize and conjugate a biotin analog
to the acceptor peptide.
[0074] The biotin ligase mutant can have various mutations,
including addition, deletion or substitution of one or more amino
acids. Preferably, the mutation will be present in the biotin
interaction and activation region, spanning amino acids 83-235.
Generally, these mutants will possess one or more amino acid
substitutions relative to the wild type biotin ligase amino acid
sequence (SEQ ID NO: 1). In most instances, the biotin ligase
mutants do not comprise an amino acid substitution (or other form
of mutation) at position 183 (which is the putative catalytic
residue) or residues near the peptide binding site and/or the ATP
binding site (amino acids 1-26).
[0075] Some mutants were developed based on an analysis of the
biotin binding site of wild type biotin ligase, particularly in the
presence of biotin. Residues that appear important in the
interaction with biotin include 89-91, 112, 115-118, 123, 186, 190,
204 and 206. Residues that influence biotin affinity include 83,
107, 115, 118, 142, 189, 207 and 235. Both types of residues are
included in the biotin interaction and activation domain. In some
important embodiments of the invention, mutants comprise amino acid
substitutions at one or more of the following positions: T90, N91,
C107, Q112, G115, R116, Y132, S134, L188, V189, I207. Specific
examples of biotin ligase mutants are proteins having at least one
of the following amino acid substitutions: T90G, T90A, T90V, C107G,
Q112M, G115A, Y132A, Y132G, S134G, V189G and I207S. The invention
contemplates the use of biotin ligase mutants having an amino acid
substitution at one or more of the afore-mentioned positions. Of
particular importance are biotin ligase mutants that harbor amino
acid substitutions at positions T90 and N91. Examples include but
are not limited to T90G/N91S, T90G/N91G, T90A/N91A, T90A/N91 L and
T90V/N91L.
[0076] The biotin ligase mutant may retain some level of activity
for biotin. Its binding affinity for biotin may be similar to that
of wild type biotin ligase. Preferably, the mutant has higher
binding affinity for a biotin analog than it does for biotin.
Consequently, biotin conjugation to an acceptor peptide would be
lower in the presence of a biotin analog. In still other
embodiments, the biotin ligase mutant has no binding affinity for
biotin.
[0077] Biotin incorporation can be measured using .sup.3H-biotin
and measuring incorporation of radioisotope in the peptide.
Conjugation of the biotin analog to an acceptor peptide can be
assayed based on inhibition of biotin incorporation. In this latter
assay, incorporation of a biotin analog is indicated by a reduced
amount of incorporated radioactivity since the biotin analog is
competed with biotin for conjugation to the acceptor peptide.
[0078] The skilled artisan will realize that conservative amino
acid substitutions may be made in biotin ligase mutants to provide
functionally equivalent variants, i.e., the variants retain the
functional capabilities of the particular biotin ligase mutant. As
used herein, a "conservative amino acid substitution" refers to an
amino acid substitution which does not alter the relative charge or
size characteristics of the protein in which the amino acid
substitution is made.
[0079] Variants can be prepared according to methods for altering
polypeptide sequence known to one of ordinary skill in the art such
as are found in references which compile such methods, e.g.
Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds.,
Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989, or Current Protocols in Molecular Biology, F.
M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York.
Conservative substitutions of amino acids include substitutions
made amongst amino acids within the following groups: (a) M, I, L,
V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g)
E, D.
[0080] Conservative amino-acid substitutions in the amino acid
sequence of biotin ligase mutants to produce functionally
equivalent variants typically are made by alteration of a nucleic
acid encoding the mutant. Such substitutions can be made by a
variety of methods known to one of ordinary skill in the art. For
example, amino acid substitutions may be made by PCR-directed
mutation, site-directed mutagenesis according to the method of
Kunkel (Kunkel, PNAS 82: 488-492, 1985), or by chemical synthesis
of a nucleic acid molecule encoding a biotin ligase mutant.
[0081] Similarly, biotin ligase mutants can be made using standard
molecular biology techniques known to those of ordinary skill in
the art. For example, the mutants may be formed by transcription
and translation from a nucleic acid sequence encoding the mutant.
Such nucleic acid sequences can be made based on the teaching of
wild type biotin ligase sequence and the position and type of amino
acid substitution.
[0082] The invention further provides methods for screening
candidate molecules for activity as a biotin ligase mutant. These
screening methods can also be combined with methods for generating
candidates. One example is a phage display library in which the
candidates can be generated and also tested for their ability to
conjugate a biotin analog to an acceptor peptide. This is
illustrated in FIG. 3 which demonstrates the use of phage having
the acceptor peptide present on their coat. Phage that display
"active" biotin ligase mutants (i.e., mutants that are able to
conjugate a biotin analog (in this case a fluorophore bearing
biotin analog) to the acceptor peptide are selected for (using an
antibody to the fluorophore). The phage can then optionally be
further manipulated to generate derivatives of the active mutant.
Phage display library technology is known in the art and has been
described extensively. (See for example Benhar, Biotechnol Adv.
2001 Feb. 1;19(1):1-33; Anthony-Cahill et al. Curr Pharm
Biotechnol. 2002 December;3(4):299-315, among others.)
[0083] The labeling methods of the invention further rely on biotin
analogs that are recognized and conjugated to acceptor peptides by
biotin ligase mutants. As used herein, a biotin analog is a
molecule that is structurally similar to biotin. (See for example
the structural similarity between ketone biotin analog, azide
biotin analog and biotin, as shown in FIG. 1B.) Biotin analogs may
share one particular structural feature in common with biotin such
as for example an aliphatic carboxylic tail, a two-ring structure,
and the like. A biotin analog may be synthesized from biotin, but
is not so limited. Examples of biotin analogs of this latter class
include biotin methyl ester, desthiobiotin, 2'-iminobiotin, and
diaminobiotin. The biotin ligase mutants must be capable of
recognizing and conjugating biotin analogs to acceptor peptides, in
a manner similar to that in which wild type biotin ligase
recognizes and conjugates biotin to the acceptor peptide.
[0084] The biotin analog binds to a biotin ligase mutant in the
interaction and activation domain. Preferably it binds with an
affinity comparable to the binding affinity of wild type biotin
ligase to biotin. However, biotin analogs that bind with lower
affinities are still useful according to the invention. In some
important embodiments, the biotin analog is not recognized by wild
type biotin ligase derived from either E. coli or from other cell
types (e.g., the cell in which the labeling reaction is
proceeding).
[0085] One category of biotin analogs are molecules having an
aliphatic carboxylic acid tail. Examples are shown in FIG. 1B.
These include but are not limited to ketone biotin analog, N-ketone
biotin analog (e.g., biotin isostere), N-alkyne biotin analog,
azide biotin analog, N-acyl azide biotin analog, N-azide biotin
analog, coumarin, fluorescein, NBD and 1,2-diamine biotin
analog.
[0086] Biotin analogs may comprise substitutions (e.g., alkylation)
at the trans-ureido nitrogen of biotin. Examples include N-ketone
biotin analog, N-alkyne biotin, N-azide and N-acyl azide, all of
which are illustrated in FIG. 1B.
[0087] Some biotin analogs are not themselves directly detectable,
while others are. In the case of the former type, the biotin analog
undergoes reaction with another moiety (either before or after
conjugation to the acceptor peptide). The subsequent modification
of this former type of biotin analog is referred to as a
bio-orthogonal ligation reaction and it is used to couple (i.e.,
label) these biotin analogs to detectable labels such as
fluorophores. The resulting moiety may be a hydrazide, phosphine,
or azide, but is not so limited. Examples of this former type of
biotin analog include ketone biotin analogs, azide biotin analogs,
N-acyl azide biotin analogs, N-azide biotin analogs, and tetrathiol
biotin analogs, among others. The structures of these biotin
analogs are illustrated in FIG. 1B.
[0088] FIG. 4 illustrates the synthesis of a ketone biotin analog.
FIG. 5 illustrates the synthesis of azide and NBD biotin analogs.
These synthesis pathways are exemplary and other synthesis
protocols can be used to generate these biotin analogs.
[0089] Accordingly, biotin analogs that are not themselves directly
detectable must be reacted with a detectable moiety. Each biotin
analog in this category will undergo a specific reaction dependent
upon its functional groups and that of its reaction partner. Some
of these reactions are shown in FIG. 2. The reaction partners in
FIG. 2 are fluorophore-bearing, however it is to be understood that
the reaction partner may comprise any detectable moiety and is not
solely limited to fluorophores. For example, a ketone biotin analog
may be reacted with a hydrazine to form a hydrazone.
Ketone-hydrazide ligation is fairly rapid and works with high
specificity on cell surfaces. (Mahal et al. Science 276:1125-1128,
1997.)
[0090] In another example, azides may be reacted with phosphines in
a Staudinger reaction. Azides and aryl phosphines generally have no
cellular counterparts. As a result, the reaction is quite specific.
Azide variants with improved stability against hydrolysis in water
at pH 6-8 are also useful in the methods of the invention. The
alkyne/azide [3+2] cycloaddition chemistry, based on Click
chemistry (Wang et al. J. Am. Chem. Soc. 125:11164-11165, 2003), is
also specific, in part because the two reactive partners do not
have cellular counterparts (i.e., the two functional groups are
non-naturally occurring).
[0091] As stated above, other biotin analogs may be themselves
directly detectable. Examples of such biotin analogs include but
are not limited to NBD-GABA, coumarin, fluorescein, Texas Red.RTM.
(sulforhodamine 101), rhodamine, rosamine, Alexa.TM. dyes,
resorufin, oregon green, tetramethyl rhodamine (TMR), carboxy
tetramethyl-rhodamine (TAMRA), Carboxy-X-rhodamine (ROX), BODIPY
dyes, and derivatives thereof. Several of these dyes are known in
the art and are commercially available (e.g., from Molecular
Probes). Several of these molecules are examples of biotin analogs
that are not derived from biotin per se. Nonetheless they share
structural similarity with biotin, making them suitable biotin
analogs for use in the methods of the invention.
[0092] The biotin analogs can also be fluorogenic. As used herein,
a fluorogenic compound is one that is not detectable (e.g.,
fluorescent) by itself, but when conjugated to another moiety
becomes fluorescent. An example of this is non-fluorescent coumarin
phosphine which reacts with azides to produce fluorescent coumarin.
Another example of a fluorogenic biotin analog is the diamine
biotin analog shown in FIG. 1B. This analog can undergo a
condensation with diaminobenzaldehyde to form a fluorescent adduct.
(Leandri et al. Gazz. Chim. Ital. 769-839, 1955.) Fluorogenic
biotin analogs are especially useful to keeping background to a
minimum (e.g., cellular imaging applications).
[0093] The invention therefore provides methods for using the
afore-mentioned biotin analogs, as well as compositions comprising
some of these analogs. For example, the invention provides
compositions comprising the NBD-GABA analog, as well as analogs
alkyated at the trans-ureido nitrogen group of biotin (e.g.,
N-ketone biotin analog, ketone biotin analog, N-alkyne biotin
analog, N-acyl azide biotin analog and N-azide biotin analog; see
FIG. 1B).
[0094] As stated above, the biotin analogs can be conjugated to
detectable labels. A "detectable label" as used herein is a
molecule or compound that can be detected by a variety of methods
including fluorescence, electrical conductivity, radioactivity,
size, and the like. The label may be of a chemical (e.g.,
carbohydrate, lipid, etc.), peptide or nucleic acid nature although
it is not so limited. The label may be directly or indirectly
detectable. The label can be detected directly for example by its
ability to emit and/or absorb light of a particular wavelength. A
label can be detected indirectly by its ability to bind, recruit
and, in some cases, cleave (or be cleaved by) another compound,
thereby emitting or absorbing energy. An example of indirect
detection is the use of an enzyme label which cleaves a substrate
into visible products.
[0095] The type of label used will depend on a variety of factors,
such as but not limited to the nature of the protein ultimately
being labeled. The label should be sterically and chemically
compatible with the biotin analog, the acceptor peptide and the
target protein. In most instances, the label should not interfere
with the activity of the target protein.
[0096] Generally, the label can be selected from the group
consisting of a fluorescent molecule, a chemiluminescent molecule
(e.g., chemiluminescent substrates), a phosphorescent molecule, a
radioisotope, an enzyme, an enzyme substrate, an affinity molecule,
a ligand, an antigen, a hapten, an antibody, an antibody fragment,
a chromogenic substrate, a contrast agent, an MRI contrast agent, a
PET label, a phosphorescent label, and the like.
[0097] Specific examples of labels include radioactive isotopes
such as .sup.32P or .sup.3H; haptens such as digoxigenin and
dintrophenyl; affinity tags such as a FLAG tag, an HA tag, a
histidine tag, a GST tag; enzyme tags such as alkaline phosphatase,
horseradish peroxidase, beta-galactosidase, etc. Other labels
include fluorophores such as fluorescein isothiocyanate ("FITC"),
Texas Red.RTM., tetramethylrhodamine isothiocyanate ("TRITC"), 4,
4-difluoro-4-bora-3a, and 4a-diaza-s-indacene ("BODIPY"), Cy-3,
Cy-5, Cy-7, Cy-Chrome.TM., R-phycoerythrin (R-PE), PerCP,
allophycocyanin (APC), PharRed.TM., Mauna Blue, Alexa.TM.350 and
other Alexa.TM. dyes, and Cascade Blue.RTM..
[0098] The labels can also be antibodies or antibody fragments or
their corresponding antigen, epitope or hapten binding partners.
Detection of such bound antibodies and proteins or peptides is
accomplished by techniques well known to those skilled in the art.
Antibody/antigen complexes which form in response to hapten
conjugates are easily detected by linking a label to the hapten or
to antibodies which recognize the hapten and then observing the
site of the label. Alternatively, the antibodies can be visualized
using secondary antibodies or fragments thereof that are specific
for the primary antibody used. Polyclonal and monoclonal antibodies
may be used. Antibody fragments include Fab, F(ab).sub.2, Fd and
antibody fragments which include a CDR3 region. The conjugates can
also be labeled using dual specificity antibodies.
[0099] The label can be a contrast agent. Contrast agents are
molecules that are administered to a subject to enhance a
particular imaging modality such as but not limited to X-ray,
ultrasound, and MRI. Examples of contrast agents for
transesophageal echocardiography (TEE) and transcranial Doppler
sonography: Echovist((R))-300 ( (TCD)); for MRI: superparamagnetic
vascular contrast agent (MION), gadolinium(III), Gd-DTPA-BMA,
superparamagnetic iron oxide (SPIO) SH U 555 A, gadoxetic acid; for
ultrasonographic (US) angiography: microbubble-based US contrast
agent (FS069); for computed tomography: iopamidol; for X-ray
venography: NC100150.
[0100] The label can be a positron emission tomography (PET) label
such as 99 m technetium and 18FDG.
[0101] The label can also be an singlet oxygen radical generator
including but not limited to resorufin, malachite green,
fluorescein, benzidine and its analogs including 2-aminobiphenyl,
4-aminobiphenyl, 3,3'-diaminobenzidine, 3,3'-dichlorobenzidine,
3,3'-dimethoxybenzidine, and 3,3'-dimethylbenzidine. These
molecules are useful in EM staining and can also be used to induce
localized toxicity.
[0102] The label can also be an analyte-binding group such as but
not limited to a metal chelator (e.g., a copper chelator). Examples
of metal chelators include EDTA, EGTA, and molecules having
pyridinium substituents, imidazole substituents, and/or thiol
substituents. These labels can be used to analyze local environment
of the target protein (e.g., Ca.sup.2+ concentration).
[0103] The label can also be a heavy atom carrier. Such labels
would be particularly useful for X-ray crystallographic study of
the target protein. Heavy atoms used in X-ray crystallography
include but are not limited to Au, Pt and Hg. An example of a heavy
atom carrier is iodine.
[0104] The label may also be a photoactivatable cross-linker. A
photoactivable cross linker is a cross linker that becomes reactive
following exposure to radiation (e.g., a ultraviolet radiation,
visible light, etc.). Examples include benzophenones, aziridines, a
photoprobe analog of geranylgeranyl diphosphate
(2-diazo-3,3,3-trifluoropropionyloxy- -farnesyl diphosphate or
DATFP-FPP) (Quellhorst et al. J Biol Chem. 2001 Nov.
2;276(44):40727-33), a DNA analogue
5-[N-(p-azidobenzoyl)-3-aminoall- yl]-dUTP (N(3)RdUTP),
sulfosuccinimidyl-2(7-azido-4-methylcoumarin-3-aceta-
mido)-ethyl-1,3'- dithiopropionate (SAED) and
1-[N-(2-hydroxy-5-azidobenzo-
yl)-2-aminoethyl]-4-(N-hydroxysuccinimidyl)-succinate.
[0105] The label may also be a photoswitch label. A photoswitch
label is a molecule that undergoes a conformational change in
response to radiation. For example, the molecule may change its
conformation from cis to trans and back again in response to
radiation. The wavelength required to induce the conformational
switch will depend upon the particular photoswitch label. Examples
of photoswitch labels include azobenzene,
3-nitro-2-naphthalenemethanol. Examples of photoswitches are also
described in van Delden et al. Chemistry. 2004 Jan. 5;10(1):61-70;
van Delden et al. Chemistry. 2003 Jun. 16;9(12): 2845-53; Zhang et
al. Bioconjug Chem. 2003 July-August;14(4):824-9; Irie et al.
Nature. 2002 Dec. 19-26;420(6917):759-60; as well as many
others.
[0106] The label may also be a photolabile protecting group.
Examples of photolabile protecting group include a nitrobenzyl
group, a dimethoxy nitrobenzyl group, nitroveratryloxycarbonyl
(NVOC), 2-(dimethylamino)-5-nitrophenyl (DANP),
Bis(o-nitrophenyl)ethanediol, brominated hydroxyquinoline, and
coumarin-4-ylmethyl derivative. Photolabile protecting groups are
useful for photocaging reactive functional groups.
[0107] The label may comprise non-naturally occurring amino acids.
Examples of non-naturally occurring amino acids include for
glutamine (Glu) or glutamic acid residues: .alpha.-aminoadipate
molecules; for tyrosine (Tyr) residues: phenylalanine (Phe),
4-carboxymethyl-Phe, pentafluoro phenylalanine (PfPhe),
4-carboxymethyl-L-phenylalanine (cmPhe),
4-carboxydifluoromethyl-L-phenylalanine (F.sub.2cmPhe),
4-phosphonomethyl-phenylalanine (Pmp),
(difluorophosphonomethyl)phenylala- nine (F.sub.2Pmp),
O-malonyl-L-tyrosine (malTyr or OMT), and fluoro-O-malonyltyrosine
(FOMT); for proline residues: 2-azetidinecarboxylic acid or
pipecolic acid (which have 6-membered, and 4-membered ring
structures respectively); 1-aminocyclohexylcarboxylic acid
(Ac.sub.6c); 3-(2-hydroxynaphtalen-1-yl)-propyl;
S-ethylisothiourea; 2-NH.sub.2-thiazoline; 2-NH.sub.2-thiazole;
asparagine residues substituted with 3-indolyl-propyl at the C
terminal carboxyl group. Modifications of cysteines, histidines,
lysines, arginines, tyrosines, glutamines, asparagines, prolines,
and carboxyl groups are known in the art and are described in U.S.
Pat. No. 6,037,134. These types of labels can be used to study
enzyme structure and function.
[0108] The label may be an enzyme or an enzyme substrate. Examples
of these include (enzyme (substrate)): Alkaline Phosphatase
(4-Methylumbelliferyl phosphate Disodium salt; 3-Phenylumbelliferyl
phosphate Hemipyridine salt); Aminopeptidase
(L-Alanine-4-methyl-7-coumar- inylamide trifluoroacetate;
Z-L-arginine-4-methyl-7-coumarinylamide hydrochloride;
Z-glycyl-L-proline-4-methyl-7-coumarinylamide); Aminopeptidase B
(L-Leucine-4-methyl-7-coumarinylamide hydrochloride);
Aminopeptidase M (L-Phenylalanine 4-methyl-7-coumarinylamide
trifluoroacetate); Butyrate esterase (4-Methylumbelliferyl
butyrate); Cellulase (2-Chloro-4-nitrophenyl-beta-D-cellobioside);
Cholinesterase (7-Acetoxy-1-methylquinolinium iodide; Resorufin
butyrate); alpha-Chymotrypsin, (Glutaryl-L-phenylalanine
4-methyl-7-coumarinylamide)- ;
N-(N-Glutaryl-L-phenylalanyl)-2-aminoacridone;
N-(N-Succinyl-L-phenylala- nyl)-2-aminoacridone); Cytochrome P450
2B6 (7-Ethoxycoumarin); Cytosolic Aldehyde Dehydrogenase (Esterase
Activity) (Resorufin acetate); Dealkylase
(O.sup.7-Pentylresorufin); Dopamine beta-hydroxylase (Tyramine);
Esterase (8-Acetoxypyrene-1,3,6-trisulfonic acid Trisodium salt;
3-(2 Benzoxazolyl)umbelliferyl acetate;
8-Butyryloxypyrene-1,3,6-tr- isulfonicacid Trisodium salt;
2',7'-Dichlorofluorescin diacetate; Fluorescein dibutyrate;
Fluorescein dilaurate; 4-Methylumbelliferyl acetate;
4-Methylumbelliferyl butyrate; 8-Octanoyloxypyrene-1,3,6-trisulf-
onic acid Trisodium salt; 8-Oleoyloxypyrene-1,3,6-trisulfonic acid
Trisodium salt; Resorufin acetate); Factor X Activated (Xa)
(4-Methylumbelliferyl 4-guanidinobenzoate hydrochloride
Monohydrate); Fucosidase, alpha-L-(
4-Methylumbelliferyl-alpha-L-fucopyranoside); Galactosidase,
alpha-(4-Methylumbelliferyl-alpha-D galactopyranoside);
Galactosidase, beta-
(6,8-Difluoro-4-methylumbelliferyl-beta-D-galactopyr- anoside;
Fluorescein di(beta-D-galactopyranoside); 4-Methylumbelliferyl-al-
pha-D-galactopyranoside; 4-Methylumbelliferyl-beta-D-lactoside:
Resorufin-beta-D-galactopyranoside;
4-(Trifluoromethyl)umbelliferyl-beta-- D-galactopyranoside;
2-Chloro-4-nitrophenyl-beta-D-lactoside); Glucosaminidase,
N-acetyl-beta- (4-Methylumbelliferyl-N-acetyl-beta-D-glu-
cosaminide Dihydrate); Glucosidase,
alpha-(4-Methylumbelliferyl-alpha-D-gl- ucopyranoside);
Glucosidase, beta- (2-Chloro-4-nitrophenyl-beta-D-glucopyr-
anoside; 6,8-Difluoro-4-methylumbelliferyl-beta-D-glucopyranoside;
4-Methylumbelliferyl-beta-D-glucopyranoside;
Resorufin-beta-D-glucopyrano- side;
4-(Trifluoromethyl)umbelliferyl-beta-D-glucopyranoside);
Glucuronidase,
beta-(6,8-Difluoro-4-methylumbelliferyl-beta-D-glucuronide Lithium
salt; 4-Methylumbelliferyl-beta-D-glucuronide Trihydrate); Leucine
aminopeptidase( L-Leucine-4-methyl-7-coumarinylamide
hydrochloride); Lipase (Fluorescein dibutyrate; Fluorescein
dilaurate; 4-Methylumbelliferyl butyrate; 4-Methylumbelliferyl
enanthate; 4-Methylumbelliferyl oleate; 4-Methylumbelliferyl
palmitate; Resorufin butyrate); Lysozyme
(4-Methylumbelliferyl-N,N',N'-triacetyl-beta-chitotri- oside);
Mannosidase, alpha- (4-Methylumbelliferyl -alpha-D-mannopyranoside-
); Monoamine oxidase (Tyramine); Monooxygenase (7-Ethoxycoumarin);
Neuraminidase (4-Methylumbelliferyl-N-acetyl-alpha-D-neuraminic
acid Sodium salt Dihydrate); Papain
(Z-L-arginine-4-methyl-7-coumarinylamide hydrochloride); Peroxidase
(Dihydrorhodamine 123); Phosphodiesterase (1-Naphthyl
4-phenylazophenyl phosphate; 2-Naphthyl 4-phenylazophenyl
phosphate); Prolyl endopeptidase
(Z-glycyl-L-proline-4-methyl-7-coumariny- lamide;
Z-glycyl-L-proline-2-naphthylamide; Z-glycyl-L-proline-4-nitroanil-
ide); Sulfatase (4-Methylumbelliferyl sulfate Potassium salt);
Thrombin (4-Methylumbelliferyl 4-guanidinobenzoate hydrochloride
Monohydrate); Trypsin (Z-L-arginine-4-methyl-7-coumarinylamide
hydrochloride; 4-Methylumbelliferyl 4-guanidinobenzoate
hydrochloride Monohydrate); Tyramine dehydrogenase (Tyramine).
[0109] It is to be understood that many of the foregoing labels can
also be biotin analogs. That is, depending upon the particular
biotin ligase mutant used, the various afore-mentioned labels may
function as biotin analogs. As such, these biotin analogs would be
considered to be directly detectable biotin analogs. In some cases,
they would not require further modification.
[0110] The labels can be attached to the biotin analogs either
before or after the analog has been conjugated to the acceptor
peptide, presuming that the label does not interfere with the
activity of biotin ligase. Labels can be attached to the biotin
analogs by any mechanism known in the art. Some of these mechanisms
are already described above for particular analogs. Other examples
of functional groups which are reactive with various labels
include, but are not limited to, (functional group: reactive group
of light emissive compound) activated ester:amines or anilines;
acyl azide:amines or anilines; acyl halide:amines, anilines,
alcohols or phenols; acyl nitrile:alcohols or phenols;
aldehyde:amines or anilines; alkyl halide:amines, anilines,
alcohols, phenols or thiols; alkyl sulfonate:thiols, alcohols or
phenols; anhydride:alcohols, phenols, amines or anilines; aryl
halide:thiols; aziridine:thiols or thioethers; carboxylic
acid:amines, anilines, alcohols or alkyl halides;
diazoalkane:carboxylic acids; epoxide:thiols; haloacetamide:thiols;
halotriazine:amines, anilines or phenols; hydrazine:aldehydes or
ketones; hydroxyamine:aldehydes or ketones; imido ester:amines or
anilines; isocyanate:amines or anilines; and isothiocyanate:amines
or anilines.
[0111] The labels are detected using a detection system. The nature
of such detection systems will depend upon the nature of the
detectable label. The detection system can be selected from any
number of detection systems known in the art. These include a
fluorescent detection system, a photographic film detection system,
a chemiluminescent detection system, an enzyme detection system, an
atomic force microscopy (AFM) detection system, a scanning
tunneling microscopy (STM) detection system, an optical detection
system, a nuclear magnetic resonance (NMR) detection system, a near
field detection system, and a total internal reflection (TIR)
detection system.
[0112] The invention provides in some instances biotin ligase
mutants and/or biotin analogs in an isolated form. As used herein,
an isolated biotin ligase mutant is a biotin ligase mutant that is
separated from its native environment in sufficiently pure form so
that it can be manipulated or used for any one of the purposes of
the invention. Thus, isolated means sufficiently pure to be used
(i) to raise and/or isolate antibodies, (ii) as a reagent in an
assay, or (iii) for sequencing, etc.
[0113] Isolated biotin analogs similarly are analogs that have been
substantially separated from either their native environment (if it
exists in nature) or their synthesis environment. Accordingly, the
biotin analogs are substantially separated from any or all reagents
present in their synthesis reaction that would be toxic or
otherwise detrimental to the target protein, the acceptor peptide,
the biotin ligase mutant, or the labeling reaction. Isolated biotin
analogs, for example, include compositions that comprise less than
25% contamination, less than 20% contamination, less than 15%
contamination, less than 10% contamination, less than 5%
contamination, or less than 1% contamination (w/w).
[0114] The invention further provides nucleic acids coding for
biotin ligase mutants. These nucleic acids therefore encode a
biotin ligase mutant having an amino acid substitution at one or
more of the following residues: 83, 89-91, 107, 112, 115-118, 123,
132, 134, 142, 186, 188, 189, 190, 204, 206, 207 and 235. In some
important embodiments, the amino acid substitution is selected from
the group consisting of T90G, T90A, T90V, C107G, Q112M, G115A,
Y132A, Y132G, S134G, V189G and l207S. Nucleic acids that encode
mutants having substitutions at two or more residues, such as
T90G/N91S, T90G/N91G, T90A/N91A, T90A/N91 L and T90V/N91L, are also
embraced by the invention.
[0115] The nucleotide sequence of wild type biotin ligase mutant is
provided as SEQ ID NO: 2. One of ordinary skill in the art will be
able to determine the codons corresponding to each of the amino
acid residues recited herein.
[0116] The invention also embraces degenerate nucleic acids that
differ from the mutant nucleic acid sequences provided herein in
codon sequence due to degeneracy of the genetic code. For example,
serine residues are encoded by the codons TCA, AGT, TCC, TCG, TCT
and AGC. Each of the six codons is equivalent for the purposes of
encoding a serine residue. Thus, it will be apparent to one of
ordinary skill in the art that any of the serine-encoding
nucleotide triplets may be employed to direct the protein synthesis
apparatus, in vitro or in vivo, to incorporate a serine residue
into an elongating mutant. Similarly, nucleotide sequence triplets
which encode other amino acid residues include, but are not limited
to: CCA, CCC, CCG and CCT (proline codons); CGA, CGC, CGG, CGT, AGA
and AGG (arginine codons); ACA, ACC, ACG and ACT (threonine
codons); AAC and AAT (asparagine codons); and ATA, ATC and ATT
(isoleucine codons). Other amino acid residues may be encoded
similarly by multiple nucleotide sequences.
[0117] The invention also involves expression vectors coding for
biotin ligase mutants and host cells containing those expression
vectors. Virtually any cells, prokaryotic or eukaryotic, which can
be transformed with heterologous DNA or RNA and which can be grown
or maintained in culture, may be used in the practice of the
invention. Examples include bacterial cells such as E. coli,
mammalian cells such as mouse, hamster, pig, goat, primate, etc.,
and other eukaryotic cells such as Xenopus cells, Drosophila cells,
Zebrafish cells, C. elegans cells, and the like. They may be of a
wide variety of tissue types, including mast cells, fibroblasts,
oocytes and lymphocytes, and they may be primary cells or cell
lines. Specific examples include CHO cells and COS cells. Cell-free
transcription systems also may be used in lieu of cells.
[0118] As used herein, a "vector" may be any of a number of nucleic
acids into which a desired sequence may be inserted by restriction
and ligation for transport between different genetic environments
or for expression in a host cell. Vectors are typically composed of
DNA although RNA vectors are also available. Vectors include, but
are not limited to, plasmids, phagemids and virus genomes. A
cloning vector is one which is able to replicate in a host cell,
and which is further characterized by one or more endonuclease
restriction sites at which the vector may be cut in a determinable
fashion and into which a desired DNA sequence may be ligated such
that the new recombinant vector retains its ability to replicate in
the host cell. In the case of plasmids, replication of the desired
sequence may occur many times as the plasmid increases in copy
number within the host bacterium or just a single time per host
before the host reproduces by mitosis. In the case of phage,
replication may occur actively during a lytic phase or passively
during a lysogenic phase.
[0119] An expression vector is one into which a desired DNA
sequence may be inserted by restriction and ligation such that it
is operably joined to regulatory sequences and may be expressed as
an RNA transcript. Vectors may further contain one or more marker
sequences (i.e., reporter sequences) suitable for use in the
identification of cells which have or have not been transformed or
transfected with the vector. Markers include, for example, genes
encoding proteins which increase or decrease either resistance or
sensitivity to antibiotics or other compounds, genes which encode
enzymes whose activities are detectable by standard assays known in
the art (e.g., beta-galactosidase or alkaline phosphatase), and
genes which visibly affect the phenotype of transformed or
transfected cells, hosts, colonies or plaques. Preferred vectors
are those capable of autonomous replication and expression of the
structural gene products present in the DNA segments to which they
are operably joined.
[0120] As used herein, a marker or coding sequence and regulatory
sequences are said to be "operably" joined when they are covalently
linked in such a way as to place the expression or transcription of
the coding sequence under the influence or control of the
regulatory sequences. If it is desired that the coding sequences be
translated into a functional protein, two DNA sequences are said to
be operably joined if induction of a promoter in the 5' regulatory
sequences results in the transcription of the coding sequence and
if the nature of the linkage between the two DNA sequences does not
(1) result in the introduction of a frame-shift mutation, (2)
interfere with the ability of the promoter region to direct the
transcription of the coding sequences, or (3) interfere with the
ability of the corresponding RNA transcript to be translated into a
protein. Thus, a promoter region would be operably joined to a
coding sequence if the promoter region were capable of effecting
transcription of that DNA sequence such that the resulting
transcript might be translated into the desired protein or
polypeptide.
[0121] The precise nature of the regulatory sequences needed for
gene expression may vary between species or cell types, but shall
in general include, as necessary, 5' non-transcribed and 5'
non-translated sequences involved with the initiation of
transcription and translation respectively, such as a TATA box,
capping sequence, CCAAT sequence, and the like. Especially, such 5'
non-transcribed regulatory sequences will include a promoter region
which includes a promoter sequence for transcriptional control of
the operably joined coding sequence. Regulatory sequences may also
include enhancer sequences or upstream activator sequences as
desired. The vectors of the invention may optionally include 5'
leader or signal sequences. The choice and design of an appropriate
vector is within the ability and discretion of one of ordinary
skill in the art.
[0122] Expression vectors containing all the necessary elements for
expression are commercially available and known to those skilled in
the art. See, e.g., Sambrook et al., Molecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory
Press, 1989. Cells are genetically engineered by the introduction
into the cells of heterologous nucleic acid, usually DNA,
molecules, encoding a biotin ligase mutant. The heterologous
nucleic acid molecules are placed under operable control of
transcriptional elements to permit the expression of the
heterologous nucleic acid molecules in the host cell.
[0123] Preferred systems for mRNA expression in mammalian cells are
those such as pcDNA3.1 (available from Invitrogen, Carlsbad,
Calif.) that contain a selectable marker such as a gene that
confers G418 resistance (which facilitates the selection of stably
transfected cell lines) and the human cytomegalovirus (CMV)
enhancer-promoter sequences. Additionally, suitable for expression
in primate or canine cell lines is the pCEP4 vector (Invitrogen,
Carlsbad, Calif.), which contains an Epstein Barr virus (EBV)
origin of replication, facilitating the maintenance of plasmid as a
multicopy extrachromosomal element. Another expression vector is
the pEF-BOS plasmid containing the promoter of polypeptide
Elongation Factor 1.alpha., which stimulates efficiently
transcription in vitro. The plasmid is described by Mishizuma and
Nagata (Nuc. Acids Res. 18:5322, 1990), and its use in transfection
experiments is disclosed by, for example, Demoulin (Mol. Cell.
Biol. 16:4710-4716, 1996). Still another preferred expression
vector is an adenovirus, described by Stratford-Perricaudet, which
is defective for E1 and E3 proteins (J. Clin. Invest. 90:626-630,
1992). The use of the adenovirus as an Adeno.P1A recombinant is
disclosed by Warnier et al., in intradermal injection in mice for
immunization against P1A (Int. J. Cancer, 67:303-310, 1996).
[0124] The invention also embraces so-called expression kits, which
allow the artisan to prepare a desired expression vector or
vectors. Such expression kits include at least separate portions of
each of the previously discussed coding sequences. Other components
may be added, as desired, as long as the previously mentioned
sequences, which are required, are included.
[0125] It will also be recognized that the invention embraces the
use of the above described, biotin ligase mutant encoding nucleic
acid containing expression vectors, to transfect host cells and
cell lines, be these prokaryotic (e.g., E. coli), or eukaryotic
(e.g., rodent cells such as CHO cells, primate cells such as COS
cells, Drosophila cells, Zebrafish cells, Xenopus cells, C. elegans
cells, yeast expression systems and recombinant baculovirus
expression in insect cells). Especially useful are mammalian cells
such as human, mouse, hamster, pig, goat, primate, etc., from a
wide variety of tissue types including primary cells and
established cell lines.
[0126] Various methods of the invention also require expression of
fusion proteins in vivo. The fusion proteins are generally
recombinantly produced proteins that comprise the biotin ligase
acceptor peptides. Such fusions can be made from virtually any
protein and those of ordinary skill in the art will be familiar
with such methods. Further conjugation methodology is also provided
in U.S. Pat. Nos. 5,932,433; 5,874,239 and 5,723,584.
[0127] In some instances, it may be desirable to place the biotin
ligase mutant and possibly the fusion protein under the control of
an inducible promoter. An inducible promoter is one that is active
in the presence (or absence) of a particular moiety. Accordingly,
it is not constitutively active. Examples of inducible promoters
are known in the art and include the tetracycline responsive
promoters and regulatory sequences such as tetracycline-inducible
T7 promoter system, and hypoxia inducible systems (Hu et al. Mol
Cell Biol. 2003 December;23(24):9361-74). Other mechanisms for
controlling expression from a particular locus include the use of
synthetic short interfering RNAs (siRNAs).
[0128] As used herein with respect to nucleic acids, the term
"isolated" means: (i) amplified in vitro by, for example,
polymerase chain reaction (PCR); (ii) recombinantly produced by
cloning; (iii) purified, as by cleavage and gel separation; or (iv)
synthesized by, for example, chemical synthesis. An isolated
nucleic acid is one which is readily manipulable by recombinant DNA
techniques well known in the art. Thus, a nucleotide sequence
contained in a vector in which 5' and 3' restriction sites are
known or for which polymerase chain reaction (PCR) primer sequences
have been disclosed is considered isolated but a nucleic acid
sequence existing in its native state in its natural host is not.
An isolated nucleic acid may be substantially purified, but need
not be. For example, a nucleic acid that is isolated within a
cloning or expression vector is not pure in that it may comprise
only a tiny percentage of the material in the cell in which it
resides. Such a nucleic acid is isolated, however, as the term is
used herein because it is readily manipulable by standard
techniques known to those of ordinary skill in the art.
[0129] As used herein, a subject shall mean an organism such as an
insect, a yeast cell, a worm, a fish, or a human or animal
including but not limited to a dog, cat, horse, cow, pig, sheep,
goat, chicken, rodent e.g., rats and mice, primate, e.g., monkey.
Subjects include vertebrate and invertebrate species. Subjects can
be house pets (e.g., dogs, cats, fish, etc.), agricultural stock
animals (e.g., cows, horses, pigs, chickens, etc.), laboratory
animals (e.g., mice, rats, rabbits, etc.), zoo animals (e.g.,
lions, giraffes, etc.), but are not so limited.
[0130] The compositions, as described above, are administered in
effective amounts for labeling of the target proteins. The
effective amount will depend upon the mode of administration, the
location of the cells being targeted, the amount of target protein
present and the level of labeling desired.
[0131] The methods of the invention, generally speaking, may be
practiced using any mode of administration that is medically
acceptable, meaning any mode that produces effective levels of the
active compounds without causing clinically unacceptable adverse
effects. A variety of administration routes are available including
but not limited to oral, rectal, topical, nasal, intradermal, or
parenteral routes. The term "parenteral" includes subcutaneous,
intravenous, intramuscular, or infusion.
[0132] When peptides are used, in certain embodiments one desirable
route of administration is by pulmonary aerosol. Techniques for
preparing aerosol delivery systems containing peptides are well
known to those of skill in the art. Generally, such systems should
utilize components which will not significantly impair the
biological properties of the peptides or proteins (see, for
example, Sciarra and Cutie, "Aerosols," in Remington's
Pharmaceutical Sciences, 18th edition, 1990, pp 1694-1712;
incorporated by reference). Those of skill in the art can readily
determine the various parameters and conditions for producing
protein or peptide aerosols without resort to undue
experimentation.
[0133] Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's or fixed oils. Intravenous vehicles include fluid
and nutrient replenishers, electrolyte replenishers (such as those
based on Ringer's dextrose), and the like. Preservatives and other
additives may also be present such as, for example, antimicrobials,
anti-oxidants, chelating agents, and inert gases and the like.
Lower doses will result from other forms of administration, such as
intravenous administration. In the event that a response in a
subject is insufficient at the initial doses applied, higher doses
(or effectively higher doses by a different, more localized
delivery route) may be employed to the extent that subject
tolerance permits. Multiple doses per day are contemplated to
achieve appropriate systemic levels of compounds.
[0134] The agents may be combined, optionally, with a
pharmaceutically-acceptable carrier. The term
"pharmaceutically-acceptabl- e carrier" as used herein means one or
more compatible solid or liquid filler, diluents or encapsulating
substances which are suitable for administration into a subject.
The term "carrier" denotes an organic or inorganic ingredient,
natural or synthetic, with which the active ingredient is combined
to facilitate the application. The components of the pharmaceutical
compositions also are capable of being commingled with the
molecules of the present invention, and with each other, in a
manner such that there is no interaction which would substantially
impair the desired pharmaceutical efficacy.
[0135] The invention in other aspects includes pharmaceutical
compositions. When administered, the pharmaceutical preparations of
the invention are applied in pharmaceutically-acceptable amounts
and in pharmaceutically-acceptably compositions. Such preparations
may routinely contain salt, buffering agents, preservatives,
compatible carriers, and the like. When used in medicine, the salts
should be pharmaceutically acceptable, but non-pharmaceutically
acceptable salts may conveniently be used to prepare
pharmaceutically-acceptable salts thereof and are not excluded from
the scope of the invention. Such pharmacologically and
pharmaceutically-acceptable salts include, but are not limited to,
those prepared from the following acids: hydrochloric, hydrobromic,
sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric,
formic, malonic, succinic, and the like. Also,
pharmaceutically-acceptable salts can be prepared as alkaline metal
or alkaline earth salts, such as sodium, potassium or calcium
salts.
[0136] Various techniques may be employed for introducing nucleic
acids of the invention into cells, depending on whether the nucleic
acids are introduced in vitro or in vivo in a host. Such techniques
include transfection of nucleic acid-CaPO.sub.4 precipitates,
transfection of nucleic acids associated with DEAE, transfection
with a retrovirus including the nucleic acid of interest, liposome
mediated transfection, and the like. For certain uses, it is
preferred to target the nucleic acid to particular cells. In such
instances, a vehicle used for delivering a nucleic acid of the
invention into a cell (e.g., a retrovirus, or other virus; a
liposome) can have a targeting molecule attached thereto. For
example, a molecule such as an antibody specific for a surface
membrane protein on the target cell or a ligand for a receptor on
the target cell can be bound to or incorporated within the nucleic
acid delivery vehicle. For example, where liposomes are employed to
deliver the nucleic acids of the invention, proteins which bind to
a surface membrane protein associated with endocytosis may be
incorporated into the liposome formulation for targeting and/or to
facilitate uptake. Such proteins include capsid proteins or
fragments thereof tropic for a particular cell type, antibodies for
proteins which undergo internalization in cycling, proteins that
target intracellular localization and enhance intracellular half
life, and the like. Polymeric delivery systems also have been used
successfully to deliver nucleic acids into cells, as is known by
those skilled in the art. Such systems even permit oral delivery of
nucleic acids.
[0137] Other delivery systems can include time-release, delayed
release or sustained release delivery systems. Such systems can
avoid repeated administrations of the labeling reagents. Many types
of release delivery systems are available and known to those of
ordinary skill in the art. They include polymer base systems such
as poly(lactide-glycolide), copolyoxalates, polycaprolactones,
polyesteramides, polyorthoesters, polyhydroxybutyric acid, and
polyanhydrides. Microcapsules of the foregoing polymers containing
drugs are described in, for example, U.S. Pat. No. 5,075,109.
Delivery systems also include non-polymer systems that are: lipids
including sterols such as cholesterol, cholesterol esters and fatty
acids or neutral fats such as mono- di- and tri-glycerides;
hydrogel release systems; sylastic systems; peptide based systems;
wax coatings; compressed tablets using conventional binders and
excipients; partially fused implants; and the like. Specific
examples include, but are not limited to: (a) erosional systems in
which the anti-inflammatory agent is contained in a form within a
matrix such as those described in U.S. Pat. Nos. 4,452,775,
4,667,014, 4,748,034 and 5,239,660 and (b) diffusional systems in
which an active component permeates at a controlled rate from a
polymer such as described in U.S. Pat. Nos. 3,832,253, and
3,854,480.
[0138] A preferred delivery system of the invention is a colloidal
dispersion system. Colloidal dispersion systems include lipid-based
systems including oil-in-water emulsions, micelles, mixed micelles,
and liposomes. A preferred colloidal system of the invention is a
liposome. Liposomes are artificial membrane vessels which are
useful as a delivery vector in vivo or in vitro. It has been shown
that large unilamellar vessels (LUV), which range in size from
0.2-4.0 .mu.m can encapsulate large macromolecules. RNA, DNA, and
intact virions can be encapsulated within the aqueous interior and
be delivered to cells in a biologically active form (Fraley, et
al., Trends Biochem. Sci., (1981) 6:77). In order for a liposome to
be an efficient gene transfer vector, one or more of the following
characteristics should be present: (1) encapsulation of the gene of
interest at high efficiency with retention of biological activity;
(2) preferential and substantial binding to a target cell in
comparison to non-target cells; (3) delivery of the aqueous
contents of the vesicle to the target cell cytoplasm at high
efficiency; and (4) accurate and effective expression of genetic
information.
[0139] Liposomes may be targeted to a particular tissue by coupling
the liposome to a specific ligand such as a monoclonal antibody,
sugar, glycolipid, or protein. Liposomes are commercially available
from Gibco BRL, for example, as LIPOFECTIN.TM. and LIPOFECTACE.TM.,
which are formed of cationic lipids such as N-[1-(2, 3
dioleyloxy)-propyl]-N, N, N-trimethylammonium chloride (DOTMA) and
dimethyl dioctadecylammonium bromide (DDAB). Methods for making
liposomes are well known in the art and have been described in many
publications. Liposomes also have been reviewed by Gregoriadis, G.
in Trends in Biotechnology, (1985) 3:235-241.
[0140] In one important embodiment, the preferred vehicle is a
biocompatible microparticle or implant that is suitable for
implantation into the mammalian recipient. Exemplary bioerodible
implants that are useful in accordance with this method are
described in PCT International application no. PCT/US/03307
(Publication No. WO 95/24929, entitled "Polymeric Gene Delivery
System"). PCT/US/03307 describes a biocompatible, preferably
biodegradable polymeric matrix for containing an exogenous gene
under the control of an appropriate promoter. The polymeric matrix
is used to achieve sustained release of the exogenous gene in the
patient. In accordance with the instant invention, the fugetactic
agents described herein are encapsulated or dispersed within the
biocompatible, preferably biodegradable polymeric matrix disclosed
in PCT/US/03307.
[0141] The polymeric matrix preferably is in the form of a
microparticle such as a microsphere (wherein an agent is dispersed
throughout a solid polymeric matrix) or a microcapsule (wherein an
agent is stored in the core of a polymeric shell). Other forms of
the polymeric matrix for containing an agent include films,
coatings, gels, implants, and stents. The size and composition of
the polymeric matrix device is selected to result in favorable
release kinetics in the tissue into which the matrix is introduced.
The size of the polymeric matrix further is selected according to
the method of delivery which is to be used. Preferably when an
aerosol route is used the polymeric matrix and agent are
encompassed in a surfactant vehicle. The polymeric matrix
composition can be selected.to have both favorable degradation
rates and also to be formed of a material which is bioadhesive, to
further increase the effectiveness of transfer. The matrix
composition also can be selected not to degrade, but rather, to
release by diffusion over an extended period of time.
[0142] In another important embodiment the delivery system is a
biocompatible microsphere that is suitable for local, site-specific
delivery. Such microspheres are disclosed in Chickering et al.,
Biotech. And Bioeng., (1996) 52:96-101 and Mathiowitz et al.,
Nature, (1997) 386:.410-414.
[0143] Both non-biodegradable and biodegradable polymeric matrices
can be used to deliver the agents of the invention to the subject.
Biodegradable matrices are preferred. Such polymers may be natural
or synthetic polymers. Synthetic polymers are preferred. The
polymer is selected based on the period of time over which release
is desired, generally in the order of a few hours to a year or
longer. Typically, release over a period ranging from between a few
hours and three to twelve months is most desirable. The polymer
optionally is in the form of a hydrogel that can absorb up to about
90% of its weight in water and further, optionally is cross-linked
with multivalent ions or other polymers.
[0144] In general, agents are delivered using a bioerodible implant
by way of diffusion, or more preferably, by degradation of the
polymeric matrix. Exemplary synthetic polymers which can be used to
form the biodegradable delivery system include: polyamides,
polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene
oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl
ethers, polyvinyl esters, poly-vinyl halides, polyvinylpyrrolidone,
polyglycolides, polysiloxanes, polyurethanes and co-polymers
thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose
ethers, cellulose esters, nitro celluloses, polymers of acrylic and
methacrylic esters, methyl cellulose, ethyl cellulose,
hydroxypropyl cellulose, hydroxy-propyl methyl cellulose,
hydroxybutyl methyl cellulose, cellulose acetate, cellulose
propionate, cellulose acetate butyrate, cellulose acetate
phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose
sulphate sodium salt, poly(methyl methacrylate), poly(ethyl
methacrylate), poly(butylmethacrylate), poly(isobutyl
methacrylate), poly(hexylmethacrylate), poly(isodecyl
methacrylate), poly(lauryl methacrylate), poly(phenyl
methacrylate), poly(methyl acrylate), poly(isopropyl acrylate),
poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene,
polypropylene, poly(ethylene glycol), poly(ethylene oxide),
poly(ethylene terephthalate), poly(vinyl alcohols), polyvinyl
acetate, poly vinyl chloride, polystyrene, polyvinylpyrrolidone,
and polymers of lactic acid and glycolic acid, polyanhydrides,
poly(ortho)esters, poly(butiric acid), poly(valeric acid), and
poly(lactide-cocaprolactone), and natural polymers such as alginate
and other polysaccharides including dextran and cellulose,
collagen, chemical derivatives thereof (substitutions, additions of
chemical groups, for example, alkyl, alkylene, hydroxylations,
oxidations, and other modifications routinely made by those skilled
in the art), albumin and other hydrophilic proteins, zein and other
prolamines and hydrophobic proteins, copolyrners and mixtures
thereof. In general, these materials degrade either by enzymatic
hydrolysis or exposure to water in vivo, by surface or bulk
erosion.
[0145] Examples of non-biodegradable polymers include ethylene
vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and
mixtures thereof.
[0146] Bioadhesive polymers of particular interest include
bioerodible hydrogels described by H. S. Sawhney, C. P. Pathak and
J. A. Hubell in Macromolecules, (1993) 26:581-587, the teachings of
which are incorporated herein, polyhyaluronic acids, casein,
gelatin, glutin, polyanhydrides, polyacrylic acid, alginate,
chitosan, poly(methyl methacrylates), poly(ethyl methacrylates),
poly(butylmethacrylate), poly(isobutyl methacrylate),
poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl
methacrylate), poly(phenyl methacrylate), poly(methyl acrylate),
poly(isopropyl acrylate), poly(isobutyl acrylate), and
poly(octadecyl acrylate).
[0147] In addition, important embodiments of the invention include
pump-based hardware delivery systems, some of which are adapted for
implantation. Such implantable pumps include controlled-release
microchips. A preferred controlled-release microchip is described
in Santini, J T Jr., et al., Nature, 1999, 397:335-338, the
contents of which are expressly incorporated herein by
reference.
[0148] Use of a long-term sustained release implant may be
particularly suitable for treatment of chronic conditions.
Long-term release, as used herein, means that the implant is
constructed and arranged to delivery therapeutic levels of the
active ingredient for at least 30 days, and preferably 60 days.
Long-term sustained release implants are well-known to those of
ordinary skill in the art and include some of the release systems
described above.
[0149] The invention will be more fully understood by reference to
the following examples. These examples, however, are merely
intended to illustrate the embodiments of the invention and are not
to be construed to limit the scope of the invention.
EXAMPLES
Introduction
[0150] Many natural enzymes have evolved marked substrate
specificity to fulfill their biological functions. One examples is
E. coli enzyme biotin ligase (i.e., BirA) which participates in the
transfer of CO.sub.2 from bicarbonate to organic acids to form
various cellular metabolite. (Chapman-Smith et al. J. Nutr.
129:477S-484S, 1999.) It has only one natural substrate in
bacteria: the biotin carboxyl carrier protein (BCCP), which it
biotinylates at lysine 122 to prepare it for carboxylation by
bicarbonate. Schatz et al. used peptide panning to identify a
minimal, 13-amino acid peptide sequence that could be recognized
and enzymatically biotinylated by BirA: LNDIFEAQKIEWH (SEQ ID
NO:4), where the biotinylated lysine is underlined. (Schatz et al.
Biotechnology 11:1138-1143, 1993; Beckett et al. Protein Sci.
8:921-929, 1999.) Purified BirA and cloning vectors for introducing
this modification sequence, called "Avi-Tag.TM.," onto proteins of
interest for site-specific biotinylation in vitro or in living
bacteria are commercially available. (Avidity, Boulder, Colo. )
Recently, Strouboulis et al. reported that BirA could also be used
to efficiently and specifically biotinylate Avi-tagged proteins in
mammalian cells. (de Boer et al. PNAS 100:7480-7485, 2003.) The E.
coli BirA does not biotinylate any endogenous mammalian proteins,
and the mammalian counterpart of BirA does not biotinylate the
Avi-Tag.
[0151] According to the invention, the biotin binding pocket of
BirA was re-engineered to accommodate a range of small-molecule
probes other than biotin. Mutants of BirA that can efficiently
catalyze the attachment of various small molecule probes (i.e.,
biotin analogs) to Avi-tagged protein substrates in vitro and in
mammalian cells have been developed. The remaining domains of the
protein were left intact, including the residues important for ATP
binding, peptide substrate binding, and catalysis. The
re-engineered BirA is useful for targeting small molecule
detectable (e.g., fluorescent) probes to specific proteins in live
cells.
[0152] i. Rational Mutation of Biotin Ligase (BirA) Active Site to
Relax its Specificity for Biotin.
[0153] The published crystallographic and biochemical data were
used to design a panel of biotin ligase mutants with altered biotin
binding sites. The two co-crystal structures of 33.5 kD BirA
complexed to biotin and biotinylated lysine show a binding pocket
composed of both hydrophobic residues (186, 204, 206) which contact
the thiophene ring of biotin, and hydrophilic residues (89, 90,
112, 115, 116, 118, 123) which form hydrogen bonds to the carbonyl
and ureido nitrogen groups. (Wilson et al. PNAS 89:9257-9261, 1992
and Weaver et al. PNAS 98:6045-6050, 2001.) Mutagenesis studies
have also identified several "second-shell" amino acids (83, 107,
142, 189, 207) important for biotin affinity.
[0154] By inspecting the 2.4 .ANG. BirA-biotin co-crystal
structure, several key residues were identified that are directly
in contact with the bicyclic core of biotin. These residues were
changed individually by mutagenesis to enlarge the biotin binding
site. Two different probes, an N-ketone biotin analog and an
N-alkyne biotin analog (FIG. 1B), were found to effectively compete
against biotin for binding to two BirA mutants--T90G and T90G/N91S,
respectively, as shown in a competitive inhibition assay using
.sup.3H-labeled biotin (Table 1). The N-ketone and N-alkyne probes
both bear substitutions on the trans ureido nitrogen of biotin,
which directly interferes with the T90 residue. Reduction of the
T90 side chain to a proton (e.g., glycine) makes room for these
ketone and alkyne moieties, allowing them to fit into the biotin
binding pocket. In the case of the alkyne probe, which has a
slightly different geometry than the ketone, additional space
generated by changing N91 to serine is required. These results show
that the BirA structure is amenable to reengineering and that
certain non-naturally occurring biotin analogs (i.e., structurally
biotin-like molecules) can be accommodated in the biotin binding
site after careful mutagenesis.
1TABLE 1 Incorporation of N-ketone and N-alkylene biotin analogsx
by the BirA mutants T90G and T90G/N91S, respectively, as measured
in a competitive inhibition assay with .sup.3H-labeled biotin. %
Inhibition of N- % Inhibition of Mutant N-Ketone .sup.3H-biotin
incorporation Mutant Alkylene .sup.3H-biotin incorporation WT 0 0%
WT 0 0% WT 4 mM <50% WT 2 mM 5% G115A 4 mM <50% Y132A 2 mM 0%
T90G/N91S 4 mM 80% G115A 2 mM 0% T90V 4 mM <50% Q112M 2 mM 1.6%
T90A 4 mM <50% T90A 2 mM 0% T90G 4 mM 100% T90A/N91A 2 mM 0%
T90A/N91L 2 mMN 0% T90V 2 mM 0% T90V/N91L 2 mM 1.6% T90G 2 mM 12%
T90G/N91S 2 mM 77%
[0155] Ketones and alkynes are useful functional groups to
incorporate into proteins because they can be subsequently ligated
in bio-orthogonal conjugation reactions to hydrazide or
azide-derivatized fluorophores. For example, specific
ketone-hydrazide ligation has been reported by Bertozzi et al. on
the surface of live mammalian cells and in cell extracts, and
alkyne-azide ligation via a [3+2] cycloaddition reaction has been
reported on Cowpea mosaic virus coat proteins and on the surface of
bacteria. (Mahal et al. Science 276:1125-1128, 1997; Wang et al. J.
Am. Chem. Soc. 125:3192-3193, 2003; Link et al. J. Am. Chem. Soc.
125:11164-11165, 2003.)
[0156] T90G has therefore been identified according to the
invention as an important residue for accommodating N-substituted
biotin analog type probes. Additional biotin analogs can be tested
for incorporation using a panel of seventeen rationally-designed
BirA point mutants: T90G, T90V, T90A, T90G/N91S, T90G/N91G,
T90A/N91A, T90A/N91L, T90V/N91L, C107G, Q112G, Q112M, G115A, Y132A,
Y132G, V189G, S143G, and I207S. Many of the contacts with biotin
are via side chains rather than backbone elements, indicating an
opportunity to carve out considerable space to accommodate
non-naturally occurring probes. Also, there is a large water-filled
channel above the ureido moiety of biotin that appears wide enough
to accommodate even larger structures (e.g., coumarin and
fluorescein).
[0157] Mutant BirA can also be expressed, purified and tested in
96-well plates. The western blot assays described herein for
analyzing probe incorporation have already been adapted to a plate
format for medium throughput.
[0158] In addition, amino acids in the biotin binding site are
being computationally randomized and subsequently analyzed using
particular algorithms to search for protein sequences that bind to
various biotin analogs with high affinity.
[0159] Biotin analog incorporation can be determined using a
variety of assays including but not limited to (1) inhibition of
.sup.3H-biotin incorporation, (2) western blot detection of
unnatural probe conjugation to cyan fluorescent protein (CFP)
bearing a C-terminal Avi-Tag, (3) MALDI mass-spectrometric
detection of probe attachment to an Avi-Tag peptide substrate, and
(4) HPLC. In the first of these assays, biotin analog candidates
and biotin are incubated together with the biotin ligase mutant and
the acceptor peptide. Decreases in incorporation of radioactivity
are indicative of a biotin analog that competes effectively with
biotin for the biotin ligase mutant activity. In the second of
these assays, biotin analog conjugation to an acceptor peptide is
indicated by the use of antibodies specific for the biotin analog
or a label conjugated thereto (e.g., an anti-FLAG antibody or an
anti-fluorophore antibody). In the third assay, differences in the
molecular weight of the acceptor peptide are indicative of
incorporation of the biotin analog. In the last of these assay,
acceptor peptides with longer retention times are indicative of
biotin analog incorporation.
[0160] ii. Synthesis of Biotin Analogs with Unique Biophysical or
Chemical Properties Such as Fluorescence.
[0161] A range of probes for both in vitro and cellular
applications was synthesized and tested against the panel of BirA
mutants. Synthesis pathways are illustrated in FIGS. 4 and 5. A
fluorophore similar in shape and size to the biotin ring system,
7-nitrobenz-2-oxa-1,3-diazole (NBD), has been conjugated to
.gamma.-aminobutyric acid (GABA) to yield NBD-GABA biotin analog
(FIG. 1B). Initial analysis of NBD-GABA indicates that it has a low
fluorescence quantum yield in water and short excitation wavelength
(.about.340 nm), making it suboptimal for live cell imaging.
However, its high sensitivity to variations in local environment
make it highly useful as an in vitro biophysical probe.
[0162] Ketone biotin analog (FIG. 1B) is not by itself a
biophysical probe, but once conjugated to a protein of interest,
can serve as a chemical handle for selective derivatization with
hydrazine or alkoxyamine-bearing probes (FIG. 2). (Cornish et al.
J. Am. Chem. Soc. 118:8150-8151, 1996; and Mahal et al. Science
276:1125-1128, 1997.) This chemistry is specific for the introduced
ketone over other functionalities present on mammalian cell
surfaces. (Mahal et al. Science 276:1125-1128, 1997.) Inside a
cell, however, hydrazides must be prevented from coupling to ketone
and aldehyde carbonyls of carbohydrates and natural cofactors. This
selectivity may be achieved through multivalency (e.g., two
modification sequences may be linked in tandem to a protein of
interest, and a bis-functionalized fluorophore with two
appropriately-spaced hydrazide groups would have a thermodynamic
preference for the target protein over endogenous carbonyl
compounds). A heterodivalent interaction may also be achieved by
introducing a cysteine residue near the lysine modification site in
the BirA target sequence and a probe bearing both a hydrazine
moiety and a thiol group would be able to form a
hydrazone-disulfide macrocyclic adduct.
[0163] Two other biotin derivatives that would similarly introduce
chemically unique handles for subsequent modification by probes are
shown in FIG. 1B. The Staudinger reaction between an azide and a
phosphine has been reported in live cells, as has complexation
between fluorescein-arsenic and a tetrathiol moiety. (Saxon et al.
Science 287:2007-2010, 2000 and Griffin et al. Science 281:269-272,
1998.)
[0164] Lastly, probes that provide readouts other than
fluorescence, or alter protein function, can also be used with the
panel of BirA mutants. Such probes may include MRI contrast
reagents, PET labels, phosphorescent or luminescent tags,
singlet-oxygen generators for electron microscopy staining, heavy
atoms, photoactivatable crosslinkers (e.g., benzophenones),
photoswitches (e.g., azobenzenes), and photocaged labels.
[0165] iii. Screening of BirA Mutants for Ability to Conjugate
Biotin Analogs to a Lysine Side Chain within a 13-Amino Acid
Consensus Sequence.
[0166] Wild-type BirA and several of the mutants listed herein have
been expressed and purified. Screening of these enzymes for ability
to conjugate NBD-GABA biotin analog to a cyan fluorescent protein
(CFP) substrate with a C-terminal 13-amino acid modification
sequence ("CFP- AviTag.TM.") is detected using anti-DNP
(dinitrophenyl) antibody (Molecular Probes) in a Western blot
format. To detect ketone conjugation, enzymatic reaction mixtures
are treated with fluorescein hydrazide, subjected to gel filtration
or Ni-NTA purification to separate CFP- AviTag.TM. (which bears an
N-terminal His.sub.6 tag) from unreacted dye, and assayed by
fluorimetry. Other biotin analogs are screened in a similar
manner.
[0167] iv. Generation of Further BirA Mutants Using a Phase Library
Approach.
[0168] Further BirA mutants can be generated using phage display
and mammalian cell FACS (fluorescence activated cell sorting). Some
of the biotin analogs described herein are sufficiently
structurally similar to biotin that they are likely to be accepted
by both wild-type BirA or one of the single-point mutants. In some
embodiments, wild type BirA may have reduced affinity for the
biotin analog however.
[0169] For other analogs, more extensive active-site reengineering
is required. Instead of screening mutants one-by-one, a more
efficient approach uses directed evolution techniques to select
suitable BirA mutants from large libraries. Neri et al. have
reported the successful display of active wild type BirA on the
surface of bacteriophage and developed an in vitro selection scheme
for separating active enzymes from inactive ones. (Heinis et al.
Protein Engineering, 14:1043-1052, 2001. ) A library of BirA
mutants was designed, using the crystal structures and biochemical
reports as guides, to be displayed on the surface of bacteriophage.
To enrich for suitable BirA mutants, anti-fluorophore antibodies
such as anti-DNP or anti-fluorescein as shown in FIG. 3A are used.
The BirA library can be DNA-shuffled between selection rounds to
increase diversity and hasten consensus towards active BirA
mutants. Negative selections against mutants still capable of
transferring biotin can also be implemented using streptavidin
beads.
[0170] A phage display-based selection system for identification of
BirA mutants capable of catalyzing biotin analog conjugation to an
Avi-Tag peptide has been developed. The selection uses a
calmodulin-M13 strategy (Heinis et al. Protein Engineering,
14:1043-1052, 2001) to anchor the Avi-Tag peptide substrate to the
protein coat of each phage molecule. The BirA library is joined to
calmodulin and this fusion protein is displayed on the phage coat
protein pIII. Model selections have demonstrated that phage
displaying wild-type BirA can be enriched over phage displaying a
dead mutant (G115S) by 42-fold in one round of selection. It has
also been shown that phage molecules chemically labeled with the
ketone probe or with the NDB probe shown above can be enriched over
mock-labeled phage by 14-fold (using antibodies against NBD or the
hydrazide-containing epitope ligated to the ketone).
[0171] Libraries that are biased for particular mutations are also
contemplated. For example, libraries that are based on a T90G amino
acid substitution are a starting template for N-substituted biotin
analogs. In other instances, the library can be randomized at seven
positions near biotin (i.e., 90, 91, 112, 115, 116, 132 and 188).
This library has a size of 1.3.times.10.sup.9.
[0172] Selection in cells is accomplished by co-transfection with a
BirA consensus substrate sequence (i.e., the acceptor peptide)
fused to cyan fluorescent protein (CFP), which displays
fluorescence resonance energy transfer (FRET) to any successfully
incorporated probe, allowing FACS selection. The advantage of
labeling an already-fluorescent protein is that non-specific
labeling of endogenous proteins will not result in a FRET signal.
Labeling specificity can be measured using the ratio of FRET to
total fluorescence.
[0173] v. In Vivo Site-specific Labeling Methodology.
[0174] BirA mutants that perform well in vitro are subsequently
screened for activity in mammalian cells. First, BirA mutants that
specifically label at the target sequence, thereby discriminating
against all endogenous mammalian proteins, are selected. E. coli
BirA has naturally evolved a significant degree of peptide
specificity in its bacterial context. Peptide panning reportedly
has shown that the substrate specificities of E. coli BirA and
yeast biotin ligase are non-overlapping. (Kiick et al. PNAS
99:19-24, 2000.) To test whether this orthogonality is also found
in the desired mammalian intracellular milieu, mammalian cells are
transfected with the BirA mutant nucleic acid sequence as described
herein and any undesired modification of endogenous mammalian
proteins is detected by Western blot. If background labeling is
observed, then the peptide substrate specificity of the enzyme will
be targeted for re-engineering using the FRET/total fluorescence
ratio readout outlined herein.
[0175] Second, biotin analogs must permeate cells and tissues
readily. Biotin is too polar to cross the plasma membrane and
requires a transporter protein. The methyl ester of biotin,
however, crosses membranes readily and is hydrolyzed to biotin
intracellularly by endogenous esterases. The membrane permeance of
biotin analogs can be tested, using fluorescence as the readout.
Probes that are too polar to cross the membrane will be derivatized
to their ester form.
[0176] Third, mutant BirA expression level must be high enough that
target proteins will be labeled efficiently. However,
overexpression can lead to toxicity. The selection strategy in some
instances would favor a stable cell line that expresses the mutant
BirA consistently and at moderate levels. Alternatively, the gene
encoding mutant BirA is placed under control of an inducible
promoter and enzyme expression is turned on only when needed.
[0177] Finally, the unconjugated probe must be washed out in order
to minimize background staining (except for fluorogenic compounds
such as FlAsH). Repeated washing with fresh growth media may be
sufficient in many cases. In others, addition of probe-specific
quenching reagents may be helpful for "stickier" small molecules.
Examples of probe-specific quenching reagents include ethandithiol
(used for example to remove unbound labels in fluorescein arsenic
labeling).
[0178] vi. Application to the Study of PI3-Kinase Activation
Patterns in 3T3-L1 Adipocytes, or Fat Storage Cells, in Response to
PDGF (Platelet-derived Growth Factor) and Insulin Stimulation.
[0179] As an example, mutant BirA can be applied to the study of
PI3-kinase activation in 3T3-L1 adipocytes. These adipocytes
display a membrane ruffling response to PDGF and a glucose
transport response to insulin, both mediated by PI3-kinase
stimulation. These differing downstream effects may result,
according to one hypothesis, from activation of spatially and/or
temporally separate pools of PI3-kinase. To test this, a two-tag
FRET system is constructed by enzymatically labeling the catalytic
and regulatory subunits of PI3-kinase inside cells. Small
fluorophores should perturb the system far less than fluorescent
proteins such as GFP. This system allows measurement of PI3-kinase
activation in real time and at subcellular resolution after insulin
or PDGF stimulation.
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Equivalents
[0199] It should be understood that the preceding is merely a
detailed description of certain embodiments. It therefore should be
apparent to those of ordinary skill in the art that various
modifications and equivalents can be made without departing from
the spirit and scope of the invention, and with no more than
routine experimentation. It is intended to encompass all such
modifications and equivalents within the scope of the appended
claims.
[0200] All references, patents and patent applications that are
recited in this application are incorporated by reference herein in
their entirety.
Sequence CWU 1
1
21 1 321 PRT Escherichia coli Bir A 1 Met Lys Asp Asn Thr Val Pro
Leu Lys Leu Ile Ala Leu Leu Ala Asn 1 5 10 15 Gly Glu Phe His Ser
Gly Glu Gln Leu Gly Glu Thr Leu Gly Met Ser 20 25 30 Arg Ala Ala
Ile Asn Lys His Ile Gln Thr Leu Arg Asp Trp Gly Val 35 40 45 Asp
Val Phe Thr Val Pro Gly Lys Gly Tyr Ser Leu Pro Glu Pro Ile 50 55
60 Gln Leu Leu Asn Ala Lys Gln Ile Leu Gly Gln Leu Asp Gly Gly Ser
65 70 75 80 Val Ala Val Leu Pro Val Ile Asp Ser Thr Asn Gln Tyr Leu
Leu Asp 85 90 95 Arg Ile Gly Glu Leu Lys Ser Gly Asp Ala Cys Ile
Ala Glu Tyr Gln 100 105 110 Gln Ala Gly Arg Gly Arg Arg Gly Arg Lys
Trp Phe Ser Pro Phe Gly 115 120 125 Ala Asn Leu Tyr Leu Ser Met Phe
Trp Arg Leu Glu Gln Gly Pro Ala 130 135 140 Ala Ala Ile Gly Leu Ser
Leu Val Ile Gly Ile Val Met Ala Glu Val 145 150 155 160 Leu Arg Lys
Leu Gly Ala Asp Lys Val Arg Val Lys Trp Pro Asn Asp 165 170 175 Leu
Tyr Leu Gln Asp Arg Lys Leu Ala Gly Ile Leu Val Glu Leu Thr 180 185
190 Gly Lys Thr Gly Asp Ala Ala Gln Ile Val Ile Gly Ala Gly Ile Asn
195 200 205 Met Ala Met Arg Arg Val Glu Glu Ser Val Val Asn Gln Gly
Trp Ile 210 215 220 Thr Leu Gln Glu Ala Gly Ile Asn Leu Asp Arg Asn
Thr Leu Ala Ala 225 230 235 240 Met Leu Ile Arg Glu Leu Arg Ala Ala
Leu Glu Leu Phe Glu Gln Glu 245 250 255 Gly Leu Ala Pro Tyr Leu Ser
Arg Trp Glu Lys Leu Asp Asn Phe Ile 260 265 270 Asn Arg Pro Val Lys
Leu Ile Ile Gly Asp Lys Glu Ile Phe Gly Ile 275 280 285 Ser Arg Gly
Ile Asp Lys Gln Gly Ala Leu Leu Leu Glu Gln Asp Gly 290 295 300 Ile
Ile Lys Pro Trp Met Gly Gly Glu Ile Ser Leu Arg Ser Ala Glu 305 310
315 320 Lys 2 966 DNA Escherichia coli Bir A 2 atgaaggata
acaccgtgcc actgaaattg attgccctgt tagcgaacgg tgaatttcac 60
tctggcgagc agttgggtga aacgctggga atgagccggg cggctattaa taaacacatt
120 cagacactgc gtgactgggg cgttgatgtc tttaccgttc cgggtaaagg
atacagcctg 180 cctgagccta tccagttact taatgctaaa cagatattgg
gtcagctgga tggcggtagt 240 gtagccgtgc tgccagtgat tgactccacg
aatcagtacc ttcttgatcg tatcggagag 300 cttaaatcgg gcgatgcttg
cattgcagaa taccagcagg ctggccgtgg tcgccggggt 360 cggaaatggt
tttcgccttt tggcgcaaac ttatatttgt cgatgttctg gcgtctggaa 420
caaggcccgg cggcggcgat tggtttaagt ctggttatcg gtatcgtgat ggcggaagta
480 ttacgcaagc tgggtgcaga taaagttcgt gttaaatggc ctaatgacct
ctatctgcag 540 gatcgcaagc tggcaggcat tctggtggag ctgactggca
aaactggcga tgcggcgcaa 600 atagtcattg gagccgggat caacatggca
atgcgccgtg ttgaagagag tgtcgttaat 660 caggggtgga tcacgctgca
ggaagcgggg atcaatctcg atcgtaatac gttggcggcc 720 atgctaatac
gtgaattacg tgctgcgttg gaactcttcg aacaagaagg attggcacct 780
tatctgtcgc gctgggaaaa gctggataat tttattaatc gcccagtgaa acttatcatt
840 ggtgataaag aaatatttgg catttcacgc ggaatagaca aacagggggc
tttattactt 900 gagcaggatg gaataataaa accctggatg ggcggtgaaa
tatccctgcg tagtgcagaa 960 aaataa 966 3 13 PRT Escherichia coli
MISC_FEATURE (2)..(2) Xaa is any amino acid 3 Leu Xaa Xaa Ile Xaa
Xaa Xaa Xaa Lys Xaa Xaa Xaa Xaa 1 5 10 4 13 PRT Artificial sequence
Oligonucleotide 4 Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp
His 1 5 10 5 15 PRT Artificial sequence Oligonucleotide 5 Gly Leu
Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu 1 5 10 15 6 321
PRT Escherichia coli 6 Met Lys Asp Asn Thr Val Pro Leu Lys Leu Ile
Ala Leu Leu Ala Asn 1 5 10 15 Gly Glu Phe His Ser Gly Glu Gln Leu
Gly Glu Thr Leu Gly Met Ser 20 25 30 Arg Ala Ala Ile Asn Lys His
Ile Gln Thr Leu Arg Asp Trp Gly Val 35 40 45 Asp Val Phe Thr Val
Pro Gly Lys Gly Tyr Ser Leu Pro Glu Pro Ile 50 55 60 Gln Leu Leu
Asn Ala Lys Gln Ile Leu Gly Gln Leu Asp Gly Gly Ser 65 70 75 80 Val
Ala Val Leu Pro Val Ile Asp Ser Gly Asn Gln Tyr Leu Leu Asp 85 90
95 Arg Ile Gly Glu Leu Lys Ser Gly Asp Ala Cys Ile Ala Glu Tyr Gln
100 105 110 Gln Ala Gly Arg Gly Arg Arg Gly Arg Lys Trp Phe Ser Pro
Phe Gly 115 120 125 Ala Asn Leu Tyr Leu Ser Met Phe Trp Arg Leu Glu
Gln Gly Pro Ala 130 135 140 Ala Ala Ile Gly Leu Ser Leu Val Ile Gly
Ile Val Met Ala Glu Val 145 150 155 160 Leu Arg Lys Leu Gly Ala Asp
Lys Val Arg Val Lys Trp Pro Asn Asp 165 170 175 Leu Tyr Leu Gln Asp
Arg Lys Leu Ala Gly Ile Leu Val Glu Leu Thr 180 185 190 Gly Lys Thr
Gly Asp Ala Ala Gln Ile Val Ile Gly Ala Gly Ile Asn 195 200 205 Met
Ala Met Arg Arg Val Glu Glu Ser Val Val Asn Gln Gly Trp Ile 210 215
220 Thr Leu Gln Glu Ala Gly Ile Asn Leu Asp Arg Asn Thr Leu Ala Ala
225 230 235 240 Met Leu Ile Arg Glu Leu Arg Ala Ala Leu Glu Leu Phe
Glu Gln Glu 245 250 255 Gly Leu Ala Pro Tyr Leu Ser Arg Trp Glu Lys
Leu Asp Asn Phe Ile 260 265 270 Asn Arg Pro Val Lys Leu Ile Ile Gly
Asp Lys Glu Ile Phe Gly Ile 275 280 285 Ser Arg Gly Ile Asp Lys Gln
Gly Ala Leu Leu Leu Glu Gln Asp Gly 290 295 300 Ile Ile Lys Pro Trp
Met Gly Gly Glu Ile Ser Leu Arg Ser Ala Glu 305 310 315 320 Lys 7
321 PRT Escherichia coli 7 Met Lys Asp Asn Thr Val Pro Leu Lys Leu
Ile Ala Leu Leu Ala Asn 1 5 10 15 Gly Glu Phe His Ser Gly Glu Gln
Leu Gly Glu Thr Leu Gly Met Ser 20 25 30 Arg Ala Ala Ile Asn Lys
His Ile Gln Thr Leu Arg Asp Trp Gly Val 35 40 45 Asp Val Phe Thr
Val Pro Gly Lys Gly Tyr Ser Leu Pro Glu Pro Ile 50 55 60 Gln Leu
Leu Asn Ala Lys Gln Ile Leu Gly Gln Leu Asp Gly Gly Ser 65 70 75 80
Val Ala Val Leu Pro Val Ile Asp Ser Gly Ser Gln Tyr Leu Leu Asp 85
90 95 Arg Ile Gly Glu Leu Lys Ser Gly Asp Ala Cys Ile Ala Glu Tyr
Gln 100 105 110 Gln Ala Gly Arg Gly Arg Arg Gly Arg Lys Trp Phe Ser
Pro Phe Gly 115 120 125 Ala Asn Leu Tyr Leu Ser Met Phe Trp Arg Leu
Glu Gln Gly Pro Ala 130 135 140 Ala Ala Ile Gly Leu Ser Leu Val Ile
Gly Ile Val Met Ala Glu Val 145 150 155 160 Leu Arg Lys Leu Gly Ala
Asp Lys Val Arg Val Lys Trp Pro Asn Asp 165 170 175 Leu Tyr Leu Gln
Asp Arg Lys Leu Ala Gly Ile Leu Val Glu Leu Thr 180 185 190 Gly Lys
Thr Gly Asp Ala Ala Gln Ile Val Ile Gly Ala Gly Ile Asn 195 200 205
Met Ala Met Arg Arg Val Glu Glu Ser Val Val Asn Gln Gly Trp Ile 210
215 220 Thr Leu Gln Glu Ala Gly Ile Asn Leu Asp Arg Asn Thr Leu Ala
Ala 225 230 235 240 Met Leu Ile Arg Glu Leu Arg Ala Ala Leu Glu Leu
Phe Glu Gln Glu 245 250 255 Gly Leu Ala Pro Tyr Leu Ser Arg Trp Glu
Lys Leu Asp Asn Phe Ile 260 265 270 Asn Arg Pro Val Lys Leu Ile Ile
Gly Asp Lys Glu Ile Phe Gly Ile 275 280 285 Ser Arg Gly Ile Asp Lys
Gln Gly Ala Leu Leu Leu Glu Gln Asp Gly 290 295 300 Ile Ile Lys Pro
Trp Met Gly Gly Glu Ile Ser Leu Arg Ser Ala Glu 305 310 315 320 Lys
8 321 PRT Escherichia coli MISC_FEATURE (83)..(83) Xaa is Val, or
any other amino acid 8 Met Lys Asp Asn Thr Val Pro Leu Lys Leu Ile
Ala Leu Leu Ala Asn 1 5 10 15 Gly Glu Phe His Ser Gly Glu Gln Leu
Gly Glu Thr Leu Gly Met Ser 20 25 30 Arg Ala Ala Ile Asn Lys His
Ile Gln Thr Leu Arg Asp Trp Gly Val 35 40 45 Asp Val Phe Thr Val
Pro Gly Lys Gly Tyr Ser Leu Pro Glu Pro Ile 50 55 60 Gln Leu Leu
Asn Ala Lys Gln Ile Leu Gly Gln Leu Asp Gly Gly Ser 65 70 75 80 Val
Ala Xaa Leu Pro Val Ile Asp Xaa Xaa Xaa Xaa Tyr Leu Leu Asp 85 90
95 Arg Ile Gly Glu Leu Lys Ser Gly Asp Ala Xaa Ile Ala Glu Tyr Xaa
100 105 110 Gln Ala Xaa Xaa Xaa Xaa Arg Gly Arg Lys Xaa Phe Ser Pro
Phe Gly 115 120 125 Ala Asn Leu Xaa Leu Xaa Met Phe Trp Arg Leu Glu
Gln Xaa Pro Ala 130 135 140 Ala Ala Ile Gly Leu Ser Leu Val Ile Gly
Ile Val Met Ala Glu Val 145 150 155 160 Leu Arg Lys Leu Gly Ala Asp
Lys Val Arg Val Lys Trp Pro Asn Asp 165 170 175 Leu Tyr Leu Gln Asp
Arg Lys Leu Ala Xaa Ile Xaa Xaa Xaa Leu Thr 180 185 190 Gly Lys Thr
Gly Asp Ala Ala Gln Ile Val Ile Xaa Ala Xaa Xaa Asn 195 200 205 Met
Ala Met Arg Arg Val Glu Glu Ser Val Val Asn Gln Gly Trp Ile 210 215
220 Thr Leu Gln Glu Ala Gly Ile Asn Leu Asp Xaa Asn Thr Leu Ala Ala
225 230 235 240 Met Leu Ile Arg Glu Leu Arg Ala Ala Leu Glu Leu Phe
Glu Gln Glu 245 250 255 Gly Leu Ala Pro Tyr Leu Ser Arg Trp Glu Lys
Leu Asp Asn Phe Ile 260 265 270 Asn Arg Pro Val Lys Leu Ile Ile Gly
Asp Lys Glu Ile Phe Gly Ile 275 280 285 Ser Arg Gly Ile Asp Lys Gln
Gly Ala Leu Leu Leu Glu Gln Asp Gly 290 295 300 Ile Ile Lys Pro Trp
Met Gly Gly Glu Ile Ser Leu Arg Ser Ala Glu 305 310 315 320 Lys 9
321 PRT Escherichia coli MISC_FEATURE (90)..(90) Xaa is Gly, Ala,
or Val 9 Met Lys Asp Asn Thr Val Pro Leu Lys Leu Ile Ala Leu Leu
Ala Asn 1 5 10 15 Gly Glu Phe His Ser Gly Glu Gln Leu Gly Glu Thr
Leu Gly Met Ser 20 25 30 Arg Ala Ala Ile Asn Lys His Ile Gln Thr
Leu Arg Asp Trp Gly Val 35 40 45 Asp Val Phe Thr Val Pro Gly Lys
Gly Tyr Ser Leu Pro Glu Pro Ile 50 55 60 Gln Leu Leu Asn Ala Lys
Gln Ile Leu Gly Gln Leu Asp Gly Gly Ser 65 70 75 80 Val Ala Val Leu
Pro Val Ile Asp Ser Xaa Asn Gln Tyr Leu Leu Asp 85 90 95 Arg Ile
Gly Glu Leu Lys Ser Gly Asp Ala Cys Ile Ala Glu Tyr Gln 100 105 110
Gln Ala Gly Arg Gly Arg Arg Gly Arg Lys Trp Phe Ser Pro Phe Gly 115
120 125 Ala Asn Leu Tyr Leu Ser Met Phe Trp Arg Leu Glu Gln Gly Pro
Ala 130 135 140 Ala Ala Ile Gly Leu Ser Leu Val Ile Gly Ile Val Met
Ala Glu Val 145 150 155 160 Leu Arg Lys Leu Gly Ala Asp Lys Val Arg
Val Lys Trp Pro Asn Asp 165 170 175 Leu Tyr Leu Gln Asp Arg Lys Leu
Ala Gly Ile Leu Val Glu Leu Thr 180 185 190 Gly Lys Thr Gly Asp Ala
Ala Gln Ile Val Ile Gly Ala Gly Ile Asn 195 200 205 Met Ala Met Arg
Arg Val Glu Glu Ser Val Val Asn Gln Gly Trp Ile 210 215 220 Thr Leu
Gln Glu Ala Gly Ile Asn Leu Asp Arg Asn Thr Leu Ala Ala 225 230 235
240 Met Leu Ile Arg Glu Leu Arg Ala Ala Leu Glu Leu Phe Glu Gln Glu
245 250 255 Gly Leu Ala Pro Tyr Leu Ser Arg Trp Glu Lys Leu Asp Asn
Phe Ile 260 265 270 Asn Arg Pro Val Lys Leu Ile Ile Gly Asp Lys Glu
Ile Phe Gly Ile 275 280 285 Ser Arg Gly Ile Asp Lys Gln Gly Ala Leu
Leu Leu Glu Gln Asp Gly 290 295 300 Ile Ile Lys Pro Trp Met Gly Gly
Glu Ile Ser Leu Arg Ser Ala Glu 305 310 315 320 Lys 10 321 PRT
Escherichia coli MISC_FEATURE (90)..(90) Xaa is Gly, Ala, or Val 10
Met Lys Asp Asn Thr Val Pro Leu Lys Leu Ile Ala Leu Leu Ala Asn 1 5
10 15 Gly Glu Phe His Ser Gly Glu Gln Leu Gly Glu Thr Leu Gly Met
Ser 20 25 30 Arg Ala Ala Ile Asn Lys His Ile Gln Thr Leu Arg Asp
Trp Gly Val 35 40 45 Asp Val Phe Thr Val Pro Gly Lys Gly Tyr Ser
Leu Pro Glu Pro Ile 50 55 60 Gln Leu Leu Asn Ala Lys Gln Ile Leu
Gly Gln Leu Asp Gly Gly Ser 65 70 75 80 Val Ala Val Leu Pro Val Ile
Asp Ser Xaa Xaa Gln Tyr Leu Leu Asp 85 90 95 Arg Ile Gly Glu Leu
Lys Ser Gly Asp Ala Cys Ile Ala Glu Tyr Gln 100 105 110 Gln Ala Gly
Arg Gly Arg Arg Gly Arg Lys Trp Phe Ser Pro Phe Gly 115 120 125 Ala
Asn Leu Tyr Leu Ser Met Phe Trp Arg Leu Glu Gln Gly Pro Ala 130 135
140 Ala Ala Ile Gly Leu Ser Leu Val Ile Gly Ile Val Met Ala Glu Val
145 150 155 160 Leu Arg Lys Leu Gly Ala Asp Lys Val Arg Val Lys Trp
Pro Asn Asp 165 170 175 Leu Tyr Leu Gln Asp Arg Lys Leu Ala Gly Ile
Leu Val Glu Leu Thr 180 185 190 Gly Lys Thr Gly Asp Ala Ala Gln Ile
Val Ile Gly Ala Gly Ile Asn 195 200 205 Met Ala Met Arg Arg Val Glu
Glu Ser Val Val Asn Gln Gly Trp Ile 210 215 220 Thr Leu Gln Glu Ala
Gly Ile Asn Leu Asp Arg Asn Thr Leu Ala Ala 225 230 235 240 Met Leu
Ile Arg Glu Leu Arg Ala Ala Leu Glu Leu Phe Glu Gln Glu 245 250 255
Gly Leu Ala Pro Tyr Leu Ser Arg Trp Glu Lys Leu Asp Asn Phe Ile 260
265 270 Asn Arg Pro Val Lys Leu Ile Ile Gly Asp Lys Glu Ile Phe Gly
Ile 275 280 285 Ser Arg Gly Ile Asp Lys Gln Gly Ala Leu Leu Leu Glu
Gln Asp Gly 290 295 300 Ile Ile Lys Pro Trp Met Gly Gly Glu Ile Ser
Leu Arg Ser Ala Glu 305 310 315 320 Lys 11 321 PRT Escherichia coli
11 Met Lys Asp Asn Thr Val Pro Leu Lys Leu Ile Ala Leu Leu Ala Asn
1 5 10 15 Gly Glu Phe His Ser Gly Glu Gln Leu Gly Glu Thr Leu Gly
Met Ser 20 25 30 Arg Ala Ala Ile Asn Lys His Ile Gln Thr Leu Arg
Asp Trp Gly Val 35 40 45 Asp Val Phe Thr Val Pro Gly Lys Gly Tyr
Ser Leu Pro Glu Pro Ile 50 55 60 Gln Leu Leu Asn Ala Lys Gln Ile
Leu Gly Gln Leu Asp Gly Gly Ser 65 70 75 80 Val Ala Val Leu Pro Val
Ile Asp Ser Gly Gly Gln Tyr Leu Leu Asp 85 90 95 Arg Ile Gly Glu
Leu Lys Ser Gly Asp Ala Cys Ile Ala Glu Tyr Gln 100 105 110 Gln Ala
Gly Arg Gly Arg Arg Gly Arg Lys Trp Phe Ser Pro Phe Gly 115 120 125
Ala Asn Leu Tyr Leu Ser Met Phe Trp Arg Leu Glu Gln Gly Pro Ala 130
135 140 Ala Ala Ile Gly Leu Ser Leu Val Ile Gly Ile Val Met Ala Glu
Val 145 150 155 160 Leu Arg Lys Leu Gly Ala Asp Lys Val Arg Val Lys
Trp Pro Asn Asp 165 170 175 Leu Tyr Leu Gln Asp Arg Lys Leu Ala Gly
Ile Leu Val Glu Leu Thr 180 185 190 Gly Lys Thr Gly Asp Ala Ala Gln
Ile Val Ile Gly Ala Gly Ile Asn 195 200 205 Met Ala Met Arg Arg Val
Glu Glu Ser Val Val Asn Gln Gly Trp Ile 210 215 220 Thr Leu Gln Glu
Ala Gly Ile Asn Leu Asp Arg Asn Thr Leu Ala Ala 225 230 235 240 Met
Leu Ile Arg Glu Leu Arg Ala Ala Leu Glu Leu
Phe Glu Gln Glu 245 250 255 Gly Leu Ala Pro Tyr Leu Ser Arg Trp Glu
Lys Leu Asp Asn Phe Ile 260 265 270 Asn Arg Pro Val Lys Leu Ile Ile
Gly Asp Lys Glu Ile Phe Gly Ile 275 280 285 Ser Arg Gly Ile Asp Lys
Gln Gly Ala Leu Leu Leu Glu Gln Asp Gly 290 295 300 Ile Ile Lys Pro
Trp Met Gly Gly Glu Ile Ser Leu Arg Ser Ala Glu 305 310 315 320 Lys
12 321 PRT Escherichia coli 12 Met Lys Asp Asn Thr Val Pro Leu Lys
Leu Ile Ala Leu Leu Ala Asn 1 5 10 15 Gly Glu Phe His Ser Gly Glu
Gln Leu Gly Glu Thr Leu Gly Met Ser 20 25 30 Arg Ala Ala Ile Asn
Lys His Ile Gln Thr Leu Arg Asp Trp Gly Val 35 40 45 Asp Val Phe
Thr Val Pro Gly Lys Gly Tyr Ser Leu Pro Glu Pro Ile 50 55 60 Gln
Leu Leu Asn Ala Lys Gln Ile Leu Gly Gln Leu Asp Gly Gly Ser 65 70
75 80 Val Ala Val Leu Pro Val Ile Asp Ser Ala Ala Gln Tyr Leu Leu
Asp 85 90 95 Arg Ile Gly Glu Leu Lys Ser Gly Asp Ala Cys Ile Ala
Glu Tyr Gln 100 105 110 Gln Ala Gly Arg Gly Arg Arg Gly Arg Lys Trp
Phe Ser Pro Phe Gly 115 120 125 Ala Asn Leu Tyr Leu Ser Met Phe Trp
Arg Leu Glu Gln Gly Pro Ala 130 135 140 Ala Ala Ile Gly Leu Ser Leu
Val Ile Gly Ile Val Met Ala Glu Val 145 150 155 160 Leu Arg Lys Leu
Gly Ala Asp Lys Val Arg Val Lys Trp Pro Asn Asp 165 170 175 Leu Tyr
Leu Gln Asp Arg Lys Leu Ala Gly Ile Leu Val Glu Leu Thr 180 185 190
Gly Lys Thr Gly Asp Ala Ala Gln Ile Val Ile Gly Ala Gly Ile Asn 195
200 205 Met Ala Met Arg Arg Val Glu Glu Ser Val Val Asn Gln Gly Trp
Ile 210 215 220 Thr Leu Gln Glu Ala Gly Ile Asn Leu Asp Arg Asn Thr
Leu Ala Ala 225 230 235 240 Met Leu Ile Arg Glu Leu Arg Ala Ala Leu
Glu Leu Phe Glu Gln Glu 245 250 255 Gly Leu Ala Pro Tyr Leu Ser Arg
Trp Glu Lys Leu Asp Asn Phe Ile 260 265 270 Asn Arg Pro Val Lys Leu
Ile Ile Gly Asp Lys Glu Ile Phe Gly Ile 275 280 285 Ser Arg Gly Ile
Asp Lys Gln Gly Ala Leu Leu Leu Glu Gln Asp Gly 290 295 300 Ile Ile
Lys Pro Trp Met Gly Gly Glu Ile Ser Leu Arg Ser Ala Glu 305 310 315
320 Lys 13 321 PRT Escherichia coli 13 Met Lys Asp Asn Thr Val Pro
Leu Lys Leu Ile Ala Leu Leu Ala Asn 1 5 10 15 Gly Glu Phe His Ser
Gly Glu Gln Leu Gly Glu Thr Leu Gly Met Ser 20 25 30 Arg Ala Ala
Ile Asn Lys His Ile Gln Thr Leu Arg Asp Trp Gly Val 35 40 45 Asp
Val Phe Thr Val Pro Gly Lys Gly Tyr Ser Leu Pro Glu Pro Ile 50 55
60 Gln Leu Leu Asn Ala Lys Gln Ile Leu Gly Gln Leu Asp Gly Gly Ser
65 70 75 80 Val Ala Val Leu Pro Val Ile Asp Ser Ala Leu Gln Tyr Leu
Leu Asp 85 90 95 Arg Ile Gly Glu Leu Lys Ser Gly Asp Ala Cys Ile
Ala Glu Tyr Gln 100 105 110 Gln Ala Gly Arg Gly Arg Arg Gly Arg Lys
Trp Phe Ser Pro Phe Gly 115 120 125 Ala Asn Leu Tyr Leu Ser Met Phe
Trp Arg Leu Glu Gln Gly Pro Ala 130 135 140 Ala Ala Ile Gly Leu Ser
Leu Val Ile Gly Ile Val Met Ala Glu Val 145 150 155 160 Leu Arg Lys
Leu Gly Ala Asp Lys Val Arg Val Lys Trp Pro Asn Asp 165 170 175 Leu
Tyr Leu Gln Asp Arg Lys Leu Ala Gly Ile Leu Val Glu Leu Thr 180 185
190 Gly Lys Thr Gly Asp Ala Ala Gln Ile Val Ile Gly Ala Gly Ile Asn
195 200 205 Met Ala Met Arg Arg Val Glu Glu Ser Val Val Asn Gln Gly
Trp Ile 210 215 220 Thr Leu Gln Glu Ala Gly Ile Asn Leu Asp Arg Asn
Thr Leu Ala Ala 225 230 235 240 Met Leu Ile Arg Glu Leu Arg Ala Ala
Leu Glu Leu Phe Glu Gln Glu 245 250 255 Gly Leu Ala Pro Tyr Leu Ser
Arg Trp Glu Lys Leu Asp Asn Phe Ile 260 265 270 Asn Arg Pro Val Lys
Leu Ile Ile Gly Asp Lys Glu Ile Phe Gly Ile 275 280 285 Ser Arg Gly
Ile Asp Lys Gln Gly Ala Leu Leu Leu Glu Gln Asp Gly 290 295 300 Ile
Ile Lys Pro Trp Met Gly Gly Glu Ile Ser Leu Arg Ser Ala Glu 305 310
315 320 Lys 14 321 PRT Escherichia coli 14 Met Lys Asp Asn Thr Val
Pro Leu Lys Leu Ile Ala Leu Leu Ala Asn 1 5 10 15 Gly Glu Phe His
Ser Gly Glu Gln Leu Gly Glu Thr Leu Gly Met Ser 20 25 30 Arg Ala
Ala Ile Asn Lys His Ile Gln Thr Leu Arg Asp Trp Gly Val 35 40 45
Asp Val Phe Thr Val Pro Gly Lys Gly Tyr Ser Leu Pro Glu Pro Ile 50
55 60 Gln Leu Leu Asn Ala Lys Gln Ile Leu Gly Gln Leu Asp Gly Gly
Ser 65 70 75 80 Val Ala Val Leu Pro Val Ile Asp Ser Thr Asn Gln Tyr
Leu Leu Asp 85 90 95 Arg Ile Gly Glu Leu Lys Ser Gly Asp Ala Gly
Ile Ala Glu Tyr Gln 100 105 110 Gln Ala Gly Arg Gly Arg Arg Gly Arg
Lys Trp Phe Ser Pro Phe Gly 115 120 125 Ala Asn Leu Tyr Leu Ser Met
Phe Trp Arg Leu Glu Gln Gly Pro Ala 130 135 140 Ala Ala Ile Gly Leu
Ser Leu Val Ile Gly Ile Val Met Ala Glu Val 145 150 155 160 Leu Arg
Lys Leu Gly Ala Asp Lys Val Arg Val Lys Trp Pro Asn Asp 165 170 175
Leu Tyr Leu Gln Asp Arg Lys Leu Ala Gly Ile Leu Val Glu Leu Thr 180
185 190 Gly Lys Thr Gly Asp Ala Ala Gln Ile Val Ile Gly Ala Gly Ile
Asn 195 200 205 Met Ala Met Arg Arg Val Glu Glu Ser Val Val Asn Gln
Gly Trp Ile 210 215 220 Thr Leu Gln Glu Ala Gly Ile Asn Leu Asp Arg
Asn Thr Leu Ala Ala 225 230 235 240 Met Leu Ile Arg Glu Leu Arg Ala
Ala Leu Glu Leu Phe Glu Gln Glu 245 250 255 Gly Leu Ala Pro Tyr Leu
Ser Arg Trp Glu Lys Leu Asp Asn Phe Ile 260 265 270 Asn Arg Pro Val
Lys Leu Ile Ile Gly Asp Lys Glu Ile Phe Gly Ile 275 280 285 Ser Arg
Gly Ile Asp Lys Gln Gly Ala Leu Leu Leu Glu Gln Asp Gly 290 295 300
Ile Ile Lys Pro Trp Met Gly Gly Glu Ile Ser Leu Arg Ser Ala Glu 305
310 315 320 Lys 15 321 PRT Escherichia coli 15 Met Lys Asp Asn Thr
Val Pro Leu Lys Leu Ile Ala Leu Leu Ala Asn 1 5 10 15 Gly Glu Phe
His Ser Gly Glu Gln Leu Gly Glu Thr Leu Gly Met Ser 20 25 30 Arg
Ala Ala Ile Asn Lys His Ile Gln Thr Leu Arg Asp Trp Gly Val 35 40
45 Asp Val Phe Thr Val Pro Gly Lys Gly Tyr Ser Leu Pro Glu Pro Ile
50 55 60 Gln Leu Leu Asn Ala Lys Gln Ile Leu Gly Gln Leu Asp Gly
Gly Ser 65 70 75 80 Val Ala Val Leu Pro Val Ile Asp Ser Thr Asn Gln
Tyr Leu Leu Asp 85 90 95 Arg Ile Gly Glu Leu Lys Ser Gly Asp Ala
Cys Ile Ala Glu Tyr Met 100 105 110 Gln Ala Gly Arg Gly Arg Arg Gly
Arg Lys Trp Phe Ser Pro Phe Gly 115 120 125 Ala Asn Leu Tyr Leu Ser
Met Phe Trp Arg Leu Glu Gln Gly Pro Ala 130 135 140 Ala Ala Ile Gly
Leu Ser Leu Val Ile Gly Ile Val Met Ala Glu Val 145 150 155 160 Leu
Arg Lys Leu Gly Ala Asp Lys Val Arg Val Lys Trp Pro Asn Asp 165 170
175 Leu Tyr Leu Gln Asp Arg Lys Leu Ala Gly Ile Leu Val Glu Leu Thr
180 185 190 Gly Lys Thr Gly Asp Ala Ala Gln Ile Val Ile Gly Ala Gly
Ile Asn 195 200 205 Met Ala Met Arg Arg Val Glu Glu Ser Val Val Asn
Gln Gly Trp Ile 210 215 220 Thr Leu Gln Glu Ala Gly Ile Asn Leu Asp
Arg Asn Thr Leu Ala Ala 225 230 235 240 Met Leu Ile Arg Glu Leu Arg
Ala Ala Leu Glu Leu Phe Glu Gln Glu 245 250 255 Gly Leu Ala Pro Tyr
Leu Ser Arg Trp Glu Lys Leu Asp Asn Phe Ile 260 265 270 Asn Arg Pro
Val Lys Leu Ile Ile Gly Asp Lys Glu Ile Phe Gly Ile 275 280 285 Ser
Arg Gly Ile Asp Lys Gln Gly Ala Leu Leu Leu Glu Gln Asp Gly 290 295
300 Ile Ile Lys Pro Trp Met Gly Gly Glu Ile Ser Leu Arg Ser Ala Glu
305 310 315 320 Lys 16 321 PRT Escherichia coli 16 Met Lys Asp Asn
Thr Val Pro Leu Lys Leu Ile Ala Leu Leu Ala Asn 1 5 10 15 Gly Glu
Phe His Ser Gly Glu Gln Leu Gly Glu Thr Leu Gly Met Ser 20 25 30
Arg Ala Ala Ile Asn Lys His Ile Gln Thr Leu Arg Asp Trp Gly Val 35
40 45 Asp Val Phe Thr Val Pro Gly Lys Gly Tyr Ser Leu Pro Glu Pro
Ile 50 55 60 Gln Leu Leu Asn Ala Lys Gln Ile Leu Gly Gln Leu Asp
Gly Gly Ser 65 70 75 80 Val Ala Val Leu Pro Val Ile Asp Ser Thr Asn
Gln Tyr Leu Leu Asp 85 90 95 Arg Ile Gly Glu Leu Lys Ser Gly Asp
Ala Cys Ile Ala Glu Tyr Gln 100 105 110 Gln Ala Ala Arg Gly Arg Arg
Gly Arg Lys Trp Phe Ser Pro Phe Gly 115 120 125 Ala Asn Leu Tyr Leu
Ser Met Phe Trp Arg Leu Glu Gln Gly Pro Ala 130 135 140 Ala Ala Ile
Gly Leu Ser Leu Val Ile Gly Ile Val Met Ala Glu Val 145 150 155 160
Leu Arg Lys Leu Gly Ala Asp Lys Val Arg Val Lys Trp Pro Asn Asp 165
170 175 Leu Tyr Leu Gln Asp Arg Lys Leu Ala Gly Ile Leu Val Glu Leu
Thr 180 185 190 Gly Lys Thr Gly Asp Ala Ala Gln Ile Val Ile Gly Ala
Gly Ile Asn 195 200 205 Met Ala Met Arg Arg Val Glu Glu Ser Val Val
Asn Gln Gly Trp Ile 210 215 220 Thr Leu Gln Glu Ala Gly Ile Asn Leu
Asp Arg Asn Thr Leu Ala Ala 225 230 235 240 Met Leu Ile Arg Glu Leu
Arg Ala Ala Leu Glu Leu Phe Glu Gln Glu 245 250 255 Gly Leu Ala Pro
Tyr Leu Ser Arg Trp Glu Lys Leu Asp Asn Phe Ile 260 265 270 Asn Arg
Pro Val Lys Leu Ile Ile Gly Asp Lys Glu Ile Phe Gly Ile 275 280 285
Ser Arg Gly Ile Asp Lys Gln Gly Ala Leu Leu Leu Glu Gln Asp Gly 290
295 300 Ile Ile Lys Pro Trp Met Gly Gly Glu Ile Ser Leu Arg Ser Ala
Glu 305 310 315 320 Lys 17 321 PRT Escherichia coli 17 Met Lys Asp
Asn Thr Val Pro Leu Lys Leu Ile Ala Leu Leu Ala Asn 1 5 10 15 Gly
Glu Phe His Ser Gly Glu Gln Leu Gly Glu Thr Leu Gly Met Ser 20 25
30 Arg Ala Ala Ile Asn Lys His Ile Gln Thr Leu Arg Asp Trp Gly Val
35 40 45 Asp Val Phe Thr Val Pro Gly Lys Gly Tyr Ser Leu Pro Glu
Pro Ile 50 55 60 Gln Leu Leu Asn Ala Lys Gln Ile Leu Gly Gln Leu
Asp Gly Gly Ser 65 70 75 80 Val Ala Val Leu Pro Val Ile Asp Ser Thr
Asn Gln Tyr Leu Leu Asp 85 90 95 Arg Ile Gly Glu Leu Lys Ser Gly
Asp Ala Cys Ile Ala Glu Tyr Gln 100 105 110 Gln Ala Gly Arg Gly Arg
Arg Gly Arg Lys Trp Phe Ser Pro Phe Gly 115 120 125 Ala Asn Leu Gly
Leu Ser Met Phe Trp Arg Leu Glu Gln Gly Pro Ala 130 135 140 Ala Ala
Ile Gly Leu Ser Leu Val Ile Gly Ile Val Met Ala Glu Val 145 150 155
160 Leu Arg Lys Leu Gly Ala Asp Lys Val Arg Val Lys Trp Pro Asn Asp
165 170 175 Leu Tyr Leu Gln Asp Arg Lys Leu Ala Gly Ile Leu Val Glu
Leu Thr 180 185 190 Gly Lys Thr Gly Asp Ala Ala Gln Ile Val Ile Gly
Ala Gly Ile Asn 195 200 205 Met Ala Met Arg Arg Val Glu Glu Ser Val
Val Asn Gln Gly Trp Ile 210 215 220 Thr Leu Gln Glu Ala Gly Ile Asn
Leu Asp Arg Asn Thr Leu Ala Ala 225 230 235 240 Met Leu Ile Arg Glu
Leu Arg Ala Ala Leu Glu Leu Phe Glu Gln Glu 245 250 255 Gly Leu Ala
Pro Tyr Leu Ser Arg Trp Glu Lys Leu Asp Asn Phe Ile 260 265 270 Asn
Arg Pro Val Lys Leu Ile Ile Gly Asp Lys Glu Ile Phe Gly Ile 275 280
285 Ser Arg Gly Ile Asp Lys Gln Gly Ala Leu Leu Leu Glu Gln Asp Gly
290 295 300 Ile Ile Lys Pro Trp Met Gly Gly Glu Ile Ser Leu Arg Ser
Ala Glu 305 310 315 320 Lys 18 321 PRT Escherichia coli 18 Met Lys
Asp Asn Thr Val Pro Leu Lys Leu Ile Ala Leu Leu Ala Asn 1 5 10 15
Gly Glu Phe His Ser Gly Glu Gln Leu Gly Glu Thr Leu Gly Met Ser 20
25 30 Arg Ala Ala Ile Asn Lys His Ile Gln Thr Leu Arg Asp Trp Gly
Val 35 40 45 Asp Val Phe Thr Val Pro Gly Lys Gly Tyr Ser Leu Pro
Glu Pro Ile 50 55 60 Gln Leu Leu Asn Ala Lys Gln Ile Leu Gly Gln
Leu Asp Gly Gly Ser 65 70 75 80 Val Ala Val Leu Pro Val Ile Asp Ser
Thr Asn Gln Tyr Leu Leu Asp 85 90 95 Arg Ile Gly Glu Leu Lys Ser
Gly Asp Ala Cys Ile Ala Glu Tyr Gln 100 105 110 Gln Ala Gly Arg Gly
Arg Arg Gly Arg Lys Trp Phe Ser Pro Phe Gly 115 120 125 Ala Asn Leu
Ala Leu Ser Met Phe Trp Arg Leu Glu Gln Gly Pro Ala 130 135 140 Ala
Ala Ile Gly Leu Ser Leu Val Ile Gly Ile Val Met Ala Glu Val 145 150
155 160 Leu Arg Lys Leu Gly Ala Asp Lys Val Arg Val Lys Trp Pro Asn
Asp 165 170 175 Leu Tyr Leu Gln Asp Arg Lys Leu Ala Gly Ile Leu Val
Glu Leu Thr 180 185 190 Gly Lys Thr Gly Asp Ala Ala Gln Ile Val Ile
Gly Ala Gly Ile Asn 195 200 205 Met Ala Met Arg Arg Val Glu Glu Ser
Val Val Asn Gln Gly Trp Ile 210 215 220 Thr Leu Gln Glu Ala Gly Ile
Asn Leu Asp Arg Asn Thr Leu Ala Ala 225 230 235 240 Met Leu Ile Arg
Glu Leu Arg Ala Ala Leu Glu Leu Phe Glu Gln Glu 245 250 255 Gly Leu
Ala Pro Tyr Leu Ser Arg Trp Glu Lys Leu Asp Asn Phe Ile 260 265 270
Asn Arg Pro Val Lys Leu Ile Ile Gly Asp Lys Glu Ile Phe Gly Ile 275
280 285 Ser Arg Gly Ile Asp Lys Gln Gly Ala Leu Leu Leu Glu Gln Asp
Gly 290 295 300 Ile Ile Lys Pro Trp Met Gly Gly Glu Ile Ser Leu Arg
Ser Ala Glu 305 310 315 320 Lys 19 321 PRT Escherichia coli 19 Met
Lys Asp Asn Thr Val Pro Leu Lys Leu Ile Ala Leu Leu Ala Asn 1 5 10
15 Gly Glu Phe His Ser Gly Glu Gln Leu Gly Glu Thr Leu Gly Met Ser
20 25 30 Arg Ala Ala Ile Asn Lys His Ile Gln Thr Leu Arg Asp Trp
Gly Val 35 40 45 Asp Val Phe Thr Val Pro Gly Lys Gly Tyr Ser Leu
Pro Glu Pro Ile 50 55 60 Gln Leu Leu Asn Ala Lys Gln Ile Leu Gly
Gln Leu Asp Gly Gly Ser 65 70 75 80 Val Ala Val Leu Pro Val Ile Asp
Ser Thr Asn Gln Tyr Leu Leu Asp 85 90 95 Arg Ile Gly Glu Leu Lys
Ser Gly Asp Ala Cys
Ile Ala Glu Tyr Gln 100 105 110 Gln Ala Gly Arg Gly Arg Arg Gly Arg
Lys Trp Phe Ser Pro Phe Gly 115 120 125 Ala Asn Leu Tyr Leu Gly Met
Phe Trp Arg Leu Glu Gln Gly Pro Ala 130 135 140 Ala Ala Ile Gly Leu
Ser Leu Val Ile Gly Ile Val Met Ala Glu Val 145 150 155 160 Leu Arg
Lys Leu Gly Ala Asp Lys Val Arg Val Lys Trp Pro Asn Asp 165 170 175
Leu Tyr Leu Gln Asp Arg Lys Leu Ala Gly Ile Leu Val Glu Leu Thr 180
185 190 Gly Lys Thr Gly Asp Ala Ala Gln Ile Val Ile Gly Ala Gly Ile
Asn 195 200 205 Met Ala Met Arg Arg Val Glu Glu Ser Val Val Asn Gln
Gly Trp Ile 210 215 220 Thr Leu Gln Glu Ala Gly Ile Asn Leu Asp Arg
Asn Thr Leu Ala Ala 225 230 235 240 Met Leu Ile Arg Glu Leu Arg Ala
Ala Leu Glu Leu Phe Glu Gln Glu 245 250 255 Gly Leu Ala Pro Tyr Leu
Ser Arg Trp Glu Lys Leu Asp Asn Phe Ile 260 265 270 Asn Arg Pro Val
Lys Leu Ile Ile Gly Asp Lys Glu Ile Phe Gly Ile 275 280 285 Ser Arg
Gly Ile Asp Lys Gln Gly Ala Leu Leu Leu Glu Gln Asp Gly 290 295 300
Ile Ile Lys Pro Trp Met Gly Gly Glu Ile Ser Leu Arg Ser Ala Glu 305
310 315 320 Lys 20 321 PRT Escherichia coli 20 Met Lys Asp Asn Thr
Val Pro Leu Lys Leu Ile Ala Leu Leu Ala Asn 1 5 10 15 Gly Glu Phe
His Ser Gly Glu Gln Leu Gly Glu Thr Leu Gly Met Ser 20 25 30 Arg
Ala Ala Ile Asn Lys His Ile Gln Thr Leu Arg Asp Trp Gly Val 35 40
45 Asp Val Phe Thr Val Pro Gly Lys Gly Tyr Ser Leu Pro Glu Pro Ile
50 55 60 Gln Leu Leu Asn Ala Lys Gln Ile Leu Gly Gln Leu Asp Gly
Gly Ser 65 70 75 80 Val Ala Val Leu Pro Val Ile Asp Ser Thr Asn Gln
Tyr Leu Leu Asp 85 90 95 Arg Ile Gly Glu Leu Lys Ser Gly Asp Ala
Cys Ile Ala Glu Tyr Gln 100 105 110 Gln Ala Gly Arg Gly Arg Arg Gly
Arg Lys Trp Phe Ser Pro Phe Gly 115 120 125 Ala Asn Leu Tyr Leu Ser
Met Phe Trp Arg Leu Glu Gln Gly Pro Ala 130 135 140 Ala Ala Ile Gly
Leu Ser Leu Val Ile Gly Ile Val Met Ala Glu Val 145 150 155 160 Leu
Arg Lys Leu Gly Ala Asp Lys Val Arg Val Lys Trp Pro Asn Asp 165 170
175 Leu Tyr Leu Gln Asp Arg Lys Leu Ala Gly Ile Leu Gly Glu Leu Thr
180 185 190 Gly Lys Thr Gly Asp Ala Ala Gln Ile Val Ile Gly Ala Gly
Ile Asn 195 200 205 Met Ala Met Arg Arg Val Glu Glu Ser Val Val Asn
Gln Gly Trp Ile 210 215 220 Thr Leu Gln Glu Ala Gly Ile Asn Leu Asp
Arg Asn Thr Leu Ala Ala 225 230 235 240 Met Leu Ile Arg Glu Leu Arg
Ala Ala Leu Glu Leu Phe Glu Gln Glu 245 250 255 Gly Leu Ala Pro Tyr
Leu Ser Arg Trp Glu Lys Leu Asp Asn Phe Ile 260 265 270 Asn Arg Pro
Val Lys Leu Ile Ile Gly Asp Lys Glu Ile Phe Gly Ile 275 280 285 Ser
Arg Gly Ile Asp Lys Gln Gly Ala Leu Leu Leu Glu Gln Asp Gly 290 295
300 Ile Ile Lys Pro Trp Met Gly Gly Glu Ile Ser Leu Arg Ser Ala Glu
305 310 315 320 Lys 21 321 PRT Escherichia coli 21 Met Lys Asp Asn
Thr Val Pro Leu Lys Leu Ile Ala Leu Leu Ala Asn 1 5 10 15 Gly Glu
Phe His Ser Gly Glu Gln Leu Gly Glu Thr Leu Gly Met Ser 20 25 30
Arg Ala Ala Ile Asn Lys His Ile Gln Thr Leu Arg Asp Trp Gly Val 35
40 45 Asp Val Phe Thr Val Pro Gly Lys Gly Tyr Ser Leu Pro Glu Pro
Ile 50 55 60 Gln Leu Leu Asn Ala Lys Gln Ile Leu Gly Gln Leu Asp
Gly Gly Ser 65 70 75 80 Val Ala Val Leu Pro Val Ile Asp Ser Thr Asn
Gln Tyr Leu Leu Asp 85 90 95 Arg Ile Gly Glu Leu Lys Ser Gly Asp
Ala Cys Ile Ala Glu Tyr Gln 100 105 110 Gln Ala Gly Arg Gly Arg Arg
Gly Arg Lys Trp Phe Ser Pro Phe Gly 115 120 125 Ala Asn Leu Tyr Leu
Ser Met Phe Trp Arg Leu Glu Gln Gly Pro Ala 130 135 140 Ala Ala Ile
Gly Leu Ser Leu Val Ile Gly Ile Val Met Ala Glu Val 145 150 155 160
Leu Arg Lys Leu Gly Ala Asp Lys Val Arg Val Lys Trp Pro Asn Asp 165
170 175 Leu Tyr Leu Gln Asp Arg Lys Leu Ala Gly Ile Leu Val Glu Leu
Thr 180 185 190 Gly Lys Thr Gly Asp Ala Ala Gln Ile Val Ile Gly Ala
Gly Ser Asn 195 200 205 Met Ala Met Arg Arg Val Glu Glu Ser Val Val
Asn Gln Gly Trp Ile 210 215 220 Thr Leu Gln Glu Ala Gly Ile Asn Leu
Asp Arg Asn Thr Leu Ala Ala 225 230 235 240 Met Leu Ile Arg Glu Leu
Arg Ala Ala Leu Glu Leu Phe Glu Gln Glu 245 250 255 Gly Leu Ala Pro
Tyr Leu Ser Arg Trp Glu Lys Leu Asp Asn Phe Ile 260 265 270 Asn Arg
Pro Val Lys Leu Ile Ile Gly Asp Lys Glu Ile Phe Gly Ile 275 280 285
Ser Arg Gly Ile Asp Lys Gln Gly Ala Leu Leu Leu Glu Gln Asp Gly 290
295 300 Ile Ile Lys Pro Trp Met Gly Gly Glu Ile Ser Leu Arg Ser Ala
Glu 305 310 315 320 Lys
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